Polymer-based antimicrobial compositions and methods of use thereof

ABSTRACT

Provided is a polymer-based antimicrobial composition that is non-toxic, water soluble, and that mitigates the transmission of infectious diseases from surfaces. The composition comprises a cationic polymer, at least one adhesion promoter, optionally organic and/or inorganic particles that are photocatalytically active in visible light, and a carrier, in which the components of the composition are not covalently bound to one another. Also provided is an antimicrobial composition that comprises at least (i) a polyethylenimine-based polymer and a carrier or (ii) an organic and/or inorganic particle that is photocatalytically active in visible light, an adhesion promoter, and a carrier. The antimicrobial compositions can be applied to disinfect a surface and to form residual self-sanitizing films on the surface that are removable.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional of U.S. patent application Ser.No. 15/662,119, filed Jul. 27, 2017, which claims the benefit of U.S.Provisional Patent Application No. 62/488,421, filed Apr. 21, 2017, andU.S. Provisional Patent Application No. 62/368,008, filed Jul. 28, 2016,each of which is incorporated by reference.

BACKGROUND OF THE INVENTION

Infectious diseases kill more people worldwide each year than any othersingle cause. Minimizing infections caused by pathogenic microorganismsis a great concern in many fields, particularly in medical devices,drugs, hospital surfaces/furniture, dental restoration and surgeryequipment, healthcare products and hygienic applications, waterpurification systems, textiles, food packaging and storage, industrialor domestic appliances, aeronautics, etc. Particularly in hospitals,great efforts and significant costs are incurred in the fight againstinfections.

Infections are produced by touching, eating, drinking, or breathingsomething that contains a pathogen. It is estimated that 80% of humaninfections occur as a result of contact with microbe-contaminatedsurfaces (Salwiczek et al., Trends Biotechnol 32: 82-90 (2014)).Generally, these infections are combated with antimicrobial agents thattarget the pathogen. Particularly problematic, however, are themicroorganisms that can rapidly and easily mutate their genes to becomeresistant to these agents, making their elimination difficult. Forinstance, Staphylococcus aureus (S. aureus) commonly colonizes humanskin and mucosa without causing severe problems, but if the bacteriaenter the body, illnesses that range from mild to life-threatening candevelop, including skin and wound infections, infected eczema, abscessinfections, heart valve infections or endocarditis, pneumonia, andbloodstream infections or bacteraemia. Some S. aureus are resistant tomethicillin and other β-lactam antibiotics—methicillin-resistant S.aureus (MRSA)—and require alternative types of antibiotics to treatthem. Moreover, the spore-forming Clostridium difficile (C. difficile),an intestinal superbug causing symptoms ranging from diarrhea tolife-threatening inflammation of the colon, is the most common bacterialinfection acquired in hospitals.

Over the past several years, there have been a growing number ofresearchers working on new antimicrobial systems aimed at helping tomitigate, combat and/or eradicate costly debilitating infections. Muchof this research has focused on polymers due to their intrinsicproperties: polymers can act as a matrix for holding antimicrobialagents and their characteristics, such as their hydrophilicity and/ormolecular weight, can have a great influence on the resultingantimicrobial activity. Therefore, the use of polymeric materials withantimicrobial properties has attracted increasing interest from both theacademic and industrial community.

Known antimicrobial polymer coatings have been prepared by impregnating,adsorbing, or covalently attaching antimicrobial agents to varioussurfaces in order to provide a filmed layer. For example: U.S. Pat. No.9,127,173 discloses preparing a layer by layer coating on a substrate,in which the coating comprises quaternary amine groups that impartantibacterial properties to the substrate. Non-leaching surfaces areoften considered preferable because microbes are exposed to high surfaceconcentrations of the antimicrobial agent compared with slow-releasesurfaces. Moreover, leaching surfaces make it difficult to pass theEnvironmental Protection Agency (EPA) cytotoxicity tests. As a generalrule, non-leaching antimicrobial coatings and preparation methodologiesare extremely complex and impractical for large-scale production andcommercialization. Furthermore, this technique is generally surfacespecific. Alternative approaches to the preparation of antimicrobialcoatings include the use of coatings that are non-covalently linked tothe surface. However, similar to the covalently linked coatings, thesemethodologies generally require complex multiple synthetic steps andneed to be adjusted for coating different substrates, thus making themimpractical for commercial use.

Therefore, despite active research in this area, there remains a needfor novel antimicrobial materials that exhibit broad-spectrumantimicrobial activity and that can easily be adapted to the complexityof different environments (e.g., homes, healthcare providers, schools,agriculture), surfaces (e.g., wood, stainless steel, marble, glass, andtextiles), and applications (e.g., food packaging, water or air filters,or even protecting fruits and vegetables). In addition, such anantimicrobial residual self-sanitizing film or coating should ideallyprovide a very high kill rate, be viable for weeks, be non-toxic yeteasily removed. It would also be desirable to have a versatile andinexpensive process for preparing such surface coatings on a commercialscale.

BRIEF SUMMARY OF THE INVENTION

The invention is predicated on a polymer-based antimicrobial compositionthat is non-toxic, water soluble, and that significantly mitigates thetransmission of infectious diseases from surfaces, such as glass,plastic, granite, and metallic substrates as well as skin. The polymersused in the compositions are capable of serving two functions: (i) theability to disinfect surfaces by killing existing germs (kill-now); and(ii) providing a removable, residual self-sanitizing film that preventsfuture microbial growth (kill-later). The polymer-based composition iseffective against bacteria, viruses, and spores, including Clostridiumdifficile (C. difficile). Additionally, unlike most commercialdisinfectants the polymer-based composition inactivates non-envelopedviruses, which typically are the cause of the common cold andgastro-intestinal flu. Because the antimicrobial composition does notrequire germicidal chemicals or metals, the composition is safe forhumans, animals, and the environment, unlike many other commercialdisinfectants.

The invention provides a polymer-based antimicrobial compositioncomprising a cationic polymer, at least one adhesion promoter, acarrier, and optionally organic and/or inorganic particles that arephotocatalytically active in visible light, wherein the components ofthe composition are not covalently bound to one another. Theantimicrobial composition is in accordance with at least one of thefollowing tests:

(i) a germicidal spray test according to American Society for Testingand Materials (ASTM) international method E1153 that meets the EPArequirement of log 3 reduction for viruses and a log 5 reduction forbacteria,

(ii) a suspension test according to ASTM international method E1052-96(2002) or ASTM international method E2315 (2016),

(iii) a film formed from the composition kills

-   -   (iii-a) at least 95% of log 5 population of a gram positive or        gram negative bacteria in 30 minutes,    -   (iii-b) at least 95% of log 4 population of an enveloped virus        within 30 minutes of contact of contact,    -   (iii-c) at least 95% of a non-enveloped virus within 30 minutes        of contact, and/or    -   (iii-d) at least 94% of a log 4 population of Clostridium        difficile bacteria within 24 hours of contact, in accordance        with Japanese Industrial Standard (JIS) Z 2801 (2006) test for        antimicrobial activity, or a modified version of such test as        described herein,

(iv) a film formed from the composition has a value of 2 or lessaccording to International Organization for Standardization (ISO)10993-5 in vitro cytotoxicity test,

(v) a durability test selected from either (v-a) a film formed from thecomposition kills at least 99.9% of gram-positive bacteria andgram-negative bacteria according to EPA Protocol #01-1A residualself-sanitizing activity test, or (v-b) waiting 7 days after filmformation, a film formed from the composition kills at least 95% ofgram-positive bacteria and gram-negative bacteria, or enveloped andnon-enveloped viruses according a modified version of Protocol #01-1Aresidual self-sanitizing activity test, as described herein.

The invention also provides a method of killing microbes on a surfacecomprising applying to the surface the antimicrobial compositioncomprising a cationic polymer, at least one adhesion promoter, acarrier, and optionally organic and/or inorganic particles that arephotocatalyically active in visible light.

The invention further provides a method of killing microbes on a surfacecomprising applying to the surface an antimicrobial compositioncomprising a high molecular weight polydiallyldimethylammonium salt anda carrier.

The invention further provides a composition comprising apolyethylenimine-based polymer, optionally a second cationic polymerselected from a polydiallyldialkylammonium salt, apoly(acrylamide-co-diallyldialkylammonium halide), chitosan, or acombination thereof, optionally a polyacid, and a carrier. Also providedis an antimicrobial composition comprising at least one organic and/orinorganic particle that is photocatalytically active in visible light,at least one adhesion promoter, and a carrier. These compositions can beused in a method of killing microbes on a surface.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates counter-ion exchange in thepolydiallyldimethylammonium chloride (polyDADMAC) by LiTFSI in anembodiment of the invention.

FIG. 2A illustrates a small pore size from a filter comprising 5 μmglass that is not positively charged. FIG. 2B illustrates a filtercomprising positively charged alumina with a larger pore size and acationic polymer coupled to the alumina.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a polymer-based antimicrobial compositioncomprising a cationic polymer, at least one adhesion promoter,optionally organic and/or inorganic particles that arephotocatalytically active in visible light, and a carrier, wherein thecomponents of the composition are not covalently bound to one another.The antimicrobial composition is in accordance with at least one of thefollowing tests:

(i) a germicidal spray test according to ASTM E1153 that meets the EPArequirement of log 3 reduction for viruses and a log 5 reduction forbacteria,

(ii) a suspension test according to ASTM E1052-96 (2002) or ASTM E2315(2016),

(iii) a film formed from the composition kills

-   -   (iii-a) at least 95% of log 5 population of a gram positive or        gram negative bacteria in 30 minutes,    -   (iii-b) at least 95% of log 4 population of an enveloped virus        within 30 minutes of contact of contact,    -   (iii-c) at least 95% of a non-enveloped virus within 30 minutes        of contact, and/or    -   (iii-d) at least 94% of a log 4 population of Clostridium        difficile bacteria within 24 hours of contact,        in accordance with JIS Z 2801 (2006) test for antimicrobial        activity, or a modified version of such test as described        herein,

(iv) a film formed from the composition has a value of 2 or lessaccording to International Organization for Standardization (ISO)10993-5 in vitro cytotoxicity test,

(v) a durability test selected from either (v-a) a film formed from thecomposition kills at least 99.9% of gram-positive bacteria andgram-negative bacteria according to EPA Protocol #01-1A residualself-sanitizing activity test, or (v-b) waiting 7 days after filmformation, a film formed from the composition kills at least 95% ofgram-positive bacteria and gram-negative bacteria, or enveloped andnon-enveloped viruses according a modified version of Protocol #01-1Aresidual self-sanitizing activity test, as described herein.

The effectiveness of the antimicrobial composition, described herein, isbest viewed in terms of the following advantages. The composition hasthe ability to “kill now” when applied to a surface as a traditionaldisinfectant—even without the presence of conventional germicidalchemicals that can be toxic. The composition has the ability to “killlater,” i.e., to kill persistently (sanitize) into the futurepost-application by forming a residual self-sanitizing film that passesan EPA-acceptable durability test and EPA-approved toxicity tests, asdescribed herein. The residual self-sanitizing film is removable withwater (e.g., warm soapy water), alcohol, or a water-alcohol mixture. Thetechnology is highly tunable because: i) the composition can be tuned tocreate films of various thicknesses, solvency, and adhesion, ii) one ormore cationic polymers can be mixed in particular proportions so as totarget specific pathogens and/or to design products with various costprofiles, and/or iii) the natural “kill-now” feature stemming from thecationic polymer can be augmented, if desired, by adding one or moreconventional antimicrobial agents to the composition. These and otheradvantages of the invention, as well as additional inventive features,will be apparent from the description of the invention provided herein.

The antimicrobial composition comprises at least one cationic polymer.The cationic polymer can be any suitable cationic polymer of a molecularweight and charge density that demonstrates an antimicrobial propertyand enables the composition or a film formed from the composition topass at least one of tests (i)-(v). It was determined that chargedensity is influenced by the molecular weight and pH of the formulation.For example, charge tends to be increased with higher molecular weight.Alternatively, or in addition, charge tends to be increased with lowerpH. Thus, the molecular weight and/or the pH can be modified to providea desired charge density and/or antimicrobial activity. Suitablemolecular weights of various cationic polymers are described herein. ThepH of the composition typically is less than about 7, such as a pHbetween about 3-7, more preferably a pH between about 4-6.

Without wishing to be bound to any particular theory, the cationicpolymer is highly effective at, inter alia, targeting gram-positiveand/or gram-negative bacteria and enveloped and non-enveloped viruses.In particular, it is believed that the positively charged polymerattracts and binds to a microbe particle, such as a virus particle. Thepolymer continues to encapsulate the microbe. Once the polymer fullyencapsulates the microbe, the capsid is destroyed, which results in aharmless release of the genomic material.

Specific examples of a suitable cationic polymer include apolydiallyldialkylammonium salt, an acryloxyalkyltrialkylammonium salt(e.g., acryloxyethyltrimethylammonium halide,methacryloxyethyltrimethylammonium halide), avinylphenalkyltrialkylammonium salt (e.g., vinylbenzyltrimethylammoniumhalide), an acrylamidoalkyltrialkylammonium salt (e.g.,3-acrylamido-3-methylbutyltrimethylammonium halide), apoly(acrylamide-co-diallyldialkylammonium salt) (e.g.,poly(acrylamide-co-diallyldimethylammonium chloride)), apolyethylenimine-based polymer, chitosan, or a combination thereof. Inany of the foregoing polymers, each alkyl group is the same or differentand is a straight chain C₁₋₆ or branched C₃₋₆ (e.g., methyl, ethyl,t-butyl) group, and the salt is an anion, such as a halide (e.g.,chloride, fluoride, bromide), a halide-containing anion (e.g.,bis(triflouromethane)sulfonimide, trifluoroacetate), a sulfate, or aphosphate. Preferably, the cationic polymer is apolydiallyldialkylammonium salt (e.g., polydiallyldimethylammoniumhalide), a poly(acrylamide-co-diallyldialkylammonium halide) (e.g.,poly(acrylamide-co-diallyldimethylammonium chloride)), and/or apolyethylenimine-based polymer (e.g., linear, non-chemically modifiedPEI). In some embodiments, the composition does not contain a bridgedpolycyclic compound (e.g., a cavitand structure), including apolymer-bound bridged polycyclic compound (e.g., a polymer-boundcavitand). In some embodiments, the cationic polymer is not a hybridmaterial that comprises one or more divalent metals and siloxanebridges.

In some instances, a combination of two or more cationic polymersselected from a polydiallyldialkylammonium salt (e.g.,polydiallyldimethylammonium halide), an acryloxyalkyltrialkylammoniumsalt, a vinylphenalkyltrialkylammonium salt, anacrylamidoalkyltrialkylammonium salt, apoly(acrylamide-co-diallyldialkylammonium halide), apolyethylenimine-based polymer, and chitosan are used in thecomposition. In a particular embodiment, a polydiallyldialkylammoniumsalt (e.g., polydiallyldimethylammonium halide) is used in combinationwith a polyethylenimine-based polymer (e.g., a linear or branchedpolyethylenimine (PEI)). In a preferred embodiment,polydiallyldimethylammonium chloride orpoly(acrylamide-co-diallyldialkylammonium chloride) is used incombination with a non-chemically modified linear PEI.

The cationic polymer may or may not be used in concert with an anionicpolymer to form a polyelectrolyte complex (PEC). As used herein, PECrefers to the complex that forms automatically upon addition of one ormore cationic polymers in concert with one or more anionic polymers. APEC typically is hydrophilic and tends to be water soluble. In someembodiments, the composition does not comprise an anionic polymer. Whenthe cationic polymer is a polydiallyldialkylammonium salt (e.g., apolydiallyldialkylammonium halide), the formation of a PEC is optional,i.e., an anionic polymer is optional in the composition. In someembodiments, the composition does not comprise an anionic polymer incombination with a polydiallyldialkylammonium salt (e.g., apolydiallydimethylammonium halide).

In an embodiment, the cationic polymer is a polydiallyldialkylammoniumsalt, such as a polydiallyldialkylammonium halide (e.g., a halide orhalide-containing anion), a polydiallyldialkylammonium sulfate, orpolydiallyldialkylammonium phosphate. In the polydiallyldialkylammoniumhalide, the halide can be any suitable compound in which the anion is ahalide or includes a halide (e.g., bis(triflouromethane)sulfonimide,trifluoroacetate), such as, polydiallyldimethylammonium fluoride,polydiallyldimethylammonium chloride, polydiallyldimethylammoniumbromide, polydiallyldimethylammonium iodide, polydiallyldimethylammoniumbis(triflouromethane)sulfonimide or a combination thereof. In preferredembodiments, the polydiallyldimethylammonium halide ispolydiallyldimethylammonium fluoride, polydiallyldimethylammoniumchloride (polyDADMAC), or a mixture of polydiallyldimethylammoniumchloride and polydiallyldimethylammonium fluoride and/orpolydiallyldimethylammonium bis(triflouromethane)sulfonimide.

Preferred polydiallyldialkylammonium salts are those polymers made frompolymerization of diallyldialkylammonium compounds, which can berepresented by the following formula:

in which R₁ and R₂ are the same or different and each is hydrogen orC₁-C₆ alkyl; R₃ and R₄ are, independently, hydrogen or an alkyl,hydroxyalkyl, carboxyalkyl, carboxyamidalkyl or alkoxyalkyl group with 1to 12 carbon atoms; and Y⁻ represents an anion such as a halide, ahalide-containing anion (e.g., bis(triflouromethane)sulfonimide), asulfate, or a phosphate. Examples of the preferred diallydialkylammoniummonomer include diallyldimethylammonium chloride (DADMAC),diallyldimethylammonium fluoride, diallyldimethylammoniumbis(triflouromethane)sulfonimide, diallyldimethylammonium bromide,diallyldimethylammonium sulfate, diallyldimethylammonium phosphate,dimethyallyldimethylammonium chloride, dimethyallyldimethylammoniumfluoride, dimethyallyldimethylammonium bis(triflouromethane)sulfonimide,diethylallyldimethylammonium chloride, diethylallyldimethylammoniumfluoride, diethylallyldimethylammonium bis(triflouromethane)sulfonimide,diallyldi(beta-hydroxyethyl)ammonium chloride,diallyldi(beta-hydroxyethyl)ammonium fluoride,diallyldi(beta-hydroxyethyl)ammonium bis(triflouromethane)sulfonimide,diallyldi(beta-ethoxyethyl)ammonium chloride,diallyldi(beta-ethoxyethyl)ammonium fluoride,diallyldi(beta-ethoxyethyl)ammonium bis(triflouromethane)sulfonimide,diallyldiethylammonium chloride, diallyldiethylammonium fluoride, anddiallyldiethylammonium bis(triflouromethane)sulfonimide. In a preferredembodiment, the cationic polymer is polyDADMAC.

In a particular embodiment, some of the polyDADMAC molecular chloridecounterions can be converted into insoluble fluoride-containingcounterions. Such conversion can occur, for example, by adding a dilutemixture of lithium bis(triflouromethane)sulfonimide (LiTFSI). LiTFSI, aswell as polyDADMAC, bears electrostatic charges that endows the moietywith a (poly)electrolyte behavior in solution. This counter-ion exchangein the polyDADMAC by LiTFSI is illustrated in FIG. 1. LiTFSI is known tohave a good solubility and stability in water. The reaction of exchangeconsists of mixing two solutions: one containing the positively chargedpolyDADMAC and the other containing the negatively charged TFSI⁻ anions.When a sufficient fraction of the polymer counter-anions has beenexchanged for TFSI⁻ anions, the polymer becomes insoluble, andprecipitates from the solution. TFSI⁻ anions in the solution either canbe linked to a polymer chain or can be part of a micelle. The presentinvention seeks to use the ion-exchange strategy to create only enoughmicelles to slightly decrease the solubility of the cationic polymerwhether used by itself or in a PEC film. The addition of the TFSI⁻anions decrease the polymer's solubility but increases the resultingfilm's durability relative to the EPA Protocol #01-1A residualself-sanitizing activity test, or a modification thereof, as describedherein. Desired solubility is achieved by experimentally determining theamount of TFSI⁻ that will yield the desired reduction in solubility. Ina specific example, the following steps can be used: 1) initially reducethe water added to the polyDADMAC solution by 125 ml; 2) create a dilutesolution of TFSI by mixing into the solution 0.125 to 0.250 grams ofTFSI for every 2.4 gram of polyDADMAC; then 3) drizzle this dilutesolution into the polyDADMAC solution. The method is carried out at roomtemperature with vigorous stirring for 24 hours, which is necessary toensure a homogeneous distribution. This mixture can, if desired, be usedto create a PEC with one or more anionic polymers. If a PEC is desiredin such an embodiment, the partial replacement of the counter ions inthe water-soluble polyDADMAC is achieved by adding a dilute solution ofTFSI⁻ before introducing an anionic polymer to create the PEC.

The counter-ion transformation strategy of polyDADMAC does not adverselyaffect its antimicrobial activity. To test the activity, excess TFSI⁻was used to create a precipitate that was then dissolved in dimethylsulfoxide (DMSO). This solution was then placed on a slide to create afilm that was held for 7 days and then inoculated with a log 6population of Escherichia coli (E. coli). The transformed polyDADMACwith a mix of fluoride and chloride ions provided a film that was ableto kill >99.99% of the E. coli population within 30 minutes.

The polydiallyldialkylammonium salt (e.g., polydiallyldimethylammoniumhalide), acryloxyalkyltrialkylammonium salt,vinylphenalkyltrialkylammonium salt, and/oracrylamidoalkyltrialkylammonium salt preferably has a number averagemolecular weight between 25,000 g/mol and 20,000,000 g/mol. A highermolecular weight typically is preferred in order to reduce thesolubility of a film formed from the antimicrobial composition. Thepolydiallyldialkylammonium salt (e.g., polydiallyldimethylammoniumhalide), acryloxyalkyltrialkylammonium salt,vinylphenalkyltrialkylammonium salt, and/oracrylamidoalkyltrialkylammonium salt can have a number average molecularweight of 20,000,000 g/mol or less, for example, 15,000,000 g/mol orless, 10,000,000 g/mol or less, 5,000,000 g/mol or less, or 1,000,000g/mol or less. Alternatively, or in addition, thepolydiallyldialkylammonium salt, acryloxyalkyltrialkylammonium salt,vinylphenalkyltrialkylammonium salt, and/oracrylamidoalkyltrialkylammonium salt can have a number average molecularweight of 25,000 g/mol or more, for example, 50,000 g/mol or more,100,000 g/mol or more, 150,000 g/mol or more, 200,000 g/mol or more,250,000 g/mol or more, 300,000 g/mol or more, 350,000 g/mol or more,400,000 g/mol or more, 450,000 g/mol or more, 500,000 g/mol or more,550,000 g/mol or more, 600,000 g/mol or more, 650,000 g/mol or more,700,000 g/mol or more, 750,000 g/mol or more, or 800,000 g/mol or more.Thus, the polydiallyldialkylammonium salt, acryloxyalkyltrialkylammoniumsalt, vinylphenalkyltrialkylammonium salt, and/oracrylamidoalkyltrialkylammonium salt can have a number average molecularweight bounded by any two of the aforementioned endpoints. For example,the polydiallyldialkylammonium salt, acryloxyalkyltrialkylammonium salt,vinylphenalkyltrialkylammonium salt, and/oracrylamidoalkyltrialkylammonium salt can have a number average molecularweight between 25,000 g/mol and 20,000,000 g/mol, between 25,000 g/moland 15,000,000 g/mol, between 25,000 g/mol and 10,000,000 g/mol, between25,000 g/mol and 5,000,000 g/mol, between 25,000 g/mol and 1,000,000g/mol, between 50,000 g/mol and 1,000,000 g/mol, between 100,000 g/moland 1,000,000 g/mol, between 150,000 g/mol and 1,000,000 g/mol, between200,000 g/mol and 1,000,000 g/mol, between 250,000 g/mol and 1,000,000g/mol, between 300,000 g/mol and 1,000,000 g/mol, between 350,000 g/moland 1,000,000 g/mol, or between 400,000 g/mol and 1,000,000 g/mol. Insome embodiments, polydiallyldialkylammonium salt,acryloxyalkyltrialkylammonium salt, vinylphenalkyltrialkylammonium salt,and/or acrylamidoalkyltrialkylammonium salt has a number averagemolecular weight between 250,000 g/mol and 1,000,000 g/mol or between800,000 g/mol and 1,000,000 g/mol, including between 900,000 g/mol and1,000,000 g/mol.

In some embodiments, the polydiallyldialkylammonium salt is “anultra-high molecular weight” polydiallyldialkylammonium salt, such as anultra high molecular weight polydiallyldimethylammonium halide. Theultra-high molecular weight polydiallyldialkylammonium salt (e.g.,polydiallyldimethylammonium halide) typically has a number averagemolecular weight between about 800,000 g/mol and about 20,000,000 g/mol(e.g., between about 1,000,000 g/mol and 15,000,000 g/mol, between about1,000,000 g/mol and 10,000,000 g/mol, between about 1,000,000 g/mol and5,000,000 g/mol, between about 2,000,000 g/mol and 5,000,000 g/mol,between about 3,000,000 g/mol and 5,000,000 g/mol, between about4,000,000 g/mol and 10,000,000 g/mol, between about 5,000,000 g/mol and20,000,000 g/mol, between about 5,000,000 g/mol and 15,000,000 g/mol,between about 6,000,000 g/mol and 20,000,000 g/mol, and between about6,000,000 g/mol and 15,000,000 g/mol). In these embodiments, generally,the halide in polydiallyldialkylammonium halide is fluoride, chloride,including anions containing fluoride and/or chloride. In particular, thepolydiallyldialkylammonium halide is polyDADMAC or a mixture ofpolyDADMAC and polydiallyldimethylammonium fluoride and/orpolydiallyldimethylammonium bis(triflouromethane)sulfonimide.

In another embodiment, the cationic polymer is a polyethylenimine-basedpolymer, which typically is effective against non-enveloped viruses,which account for a large number of pathogenic microbes, such asrhinovirus, poliovirus, adenoviruses, coxsackievirus, parvovirus, androtavirus. The polyethylenimine-based polymer can be any suitablepolyethylenimine-based polymer that is linear or non-linear, preferablylinear.

There are a number of reports of polyethylenimine (PEI) that has beenchemically modified to create an antimicrobial agent. See, for example,Gao et al. (J. Biomaterial Science, Polymer Edition, 2007, 18, 531-544)reported that quaternized branched PEI (BPEI) was antimicrobial againstEscherichia coli (E. coli) at low concentrations. Pasquier et al.(Biomacromolecules, 2007, 8, 2874-2882) reported that BPEI quaternizedwith various long alkyl groups exhibited some degree of antimicrobialactivity against E. coli, while linear PEI (LPEI) grafted with longalkyl chains produced a series of hydrophobically-modified waterinsoluble LPEI derivatives that effectively killed E. coli andStaphyloccoccus aureus. See also, U.S. Pat. No. 9,399,044 and WO2008/127416 A2. For example, the chemically modified PEI described inU.S. Pat. No. 9,399,044 is only effective against bacterium (e.g.,Tuberculosis mycobacterium, gram negative E. coli and Pseudomonasaeruginosa, gram positive Staphylococcus aureus) and the fungus Candidaalbicans, but not viruses. WO 2008/127416 A2 demonstrates that anantimicrobial coating comprising a chemically modified PEI is capable ofkilling enveloped viruses but not non-enveloped viruses, as shown inTable 1.

TABLE 1 Final Viral Titer Initial Viral (pfu/ml) Titer Coated VirusTiter Strain (pfu/ml) Uncoated slide slide Reduction A/Wuhan/ (4.8 ±0.5) × (3.1 ± 0.4) × 0 100% 359/95 10⁵ 10³ (>3.5 logs) A/turkey/ (6.1 ±1.1) × (3.7 ± 0.4) × 0 100% MN/833/80 10⁶ 10⁴ (>4.5 logs)

However, chemical modification requires expensive, low yield, organicchemical processes that use toxic chemicals that are harmful to humansand the environment. Thus, in some embodiments of the invention, thepolyethylenimine-based polymer is linear PEI that has not beenchemically or structurally modified (e.g., does not include alkyl and/orquaternary ammonium groups). Moreover, it was discovered thatnon-chemically modified linear PEI can kill non-enveloped viruses. Inparticular, a film of the non-chemically modified linear PEI describedherein kills not only gram positive and gram negative bacteria but alsodemonstrates at least a log 4 (99.99%) reduction against both envelopedand non-enveloped viruses, which is particularly important because manynon-enveloped viruses are the pathogenic microbes that cause commoncolds and gastrointestinal flu, such as rhinovirus, poliovirus,adenoviruses, coxsackievirus, parvovirus, and rotavirus. The ability ofan antimicrobial composition comprising non-chemically modified, linearPEI to reduce an MS2 bacteriophage, which is considered to be asurrogate for a non-enveloped virus, is demonstrated in Table 2.

TABLE 2 Percent Log₁₀ Reduction Reduction Compared Compared Micro-Contact Test PFU/ to Parallel to Parallel organism Time SubstanceCarrier Control Control MS2 Initial Inoculum 4.00E+05 n/a bacteriophage5 min Parallel 1.60E+05 ATCC control 15597-B1 Non- 1.00E+01 99.994% 4.20chemically modified, linear PEI, 6K ppm, pH 6.4

In other embodiments, the polyethylenimine-based polymer is a deacylatedPEI or a quaternized N-alkyl-N-methylpolyethylenimine. The deacylatedpolyethylenimine can be supplied by a commercial source, such asPolysciences, Inc. (Warrington, Pa.). As used herein, “deacylatedpolyethylenimine” refers to a polyethylenimine with protonatablenitrogens and of the formula:

wherein the polymer has been partially (at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, or at least 90%) or fully (about 98-100%) hydrolyzed(deacylated). Without wishing to be bound to any particular theory, itis believed that dealkylation strengthens PEI's ability to kill virusesand decreases its cytotoxicity.

As used herein, “quaternized-N-alkyl-N-methylpolyethylenimine” refers toa polyethylenimine that has been partially (at least 10%, at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, or at least 90%) or fully (about 98-100%) hydrolyzed,methylated, then quaternized with an alkyl substituent. The alkylsubstituent in this embodiment can be any suitable alkyl substituentthat is straight chain or branched. Generally, the alkyl substituent hasa chain length chosen to be most effective against viruses, e.g., C₁₋₁₈,including C₈₋₁₄ and C₁₀₋₁₂. In an embodiment, the alkyl substituent isdecane, dodecane, or hexadecane.

A synthetic route to providing a PEI that is fully hydrolyzed(deacylated), methylated, and then quaternized includes the followingmethod steps:

Step 1: Prepare a fully deacetylated linear PEI by the acid-catalyzedhydrolysis of commercial PEOZs (e.g., 500 kDa, 200 kDa, and 50 kDa,preferably 50 kDa). For example, 10.0 g of the PEOZs was added to 400 mLof 24% (wt/vol) HCl, followed by refluxing for 96 h. The POEZ crystalsdissolved completely in 2h, but 3h later, (i.e., total of 5h) a whiteprecipitate appeared. The precipitate in each case was isolated byfiltration and then air-dried.

The protonated PEOZ (2-ethyl-2-oxazoline) was then deprotonated using anaqueous base (e.g., KOH) solution. Briefly, 10 g of protonated linearPEI was dissolved in distilled water (50 mL), and 6 M KOH was addeduntil the pH of the solution became ˜11. The fully deprotonated PEIsappeared as a white precipitate, which was filtered and washedrepeatedly with distilled water until it became neutral (pH ˜7). The endproduct was a linear PEI without N-acyl groups having a molecular weightof approximately 217 kDa, 87 kDa, or preferably 22 kDa.

Step 2: The E. Clarke methylation technique (Clarke et al., JAGS,55(11): 4571 (1933)) can be used to create linear N-Methyl-PEI. A 50%aqueous solution of PEI consisting of 10 g of 22 kDa PEI created in step1 was transferred to a round-bottom flask to which 90% formic acid (24.5mL, 0.48 mol) was added followed by 37% formaldehyde (29.3 mL, 0.36 mol)and 20 mL of water. The reaction mixture was stirred at 90° C. for 96h.After cooling to room temperature, the pH of the reaction mixture wasadjusted to 11 using an 8M KOH solution. The deprotonated N-methylatedPEI was extracted several times with chloroform, and the entire organicsolution was subjected to repeated water wash. Chloroform was thenremoved to yield a yellow viscous N-methylated PEI with 100% degree ofmethylation.

Step 3: N-Alkyl N-Methyl PEI was then quaternized to be water solubleand target viruses. In particular, lg (17.5 mmol/repeating unit) ofN-methylated PEI was dissolved in 75 ml tert-butanol in a screw-toppressure tube. To this was added 1-bromohexadecane, to provide the sidechain lengths that are most effective against viruses. The reactionmixture then was heated at 105° C. for 48h to 96h, depending upon thedesired solubility of the end product. After completing the reaction,the solvent was removed to one-tenth of its initial volume. Then, anexcess of acetone (200 mL) was added to the reaction mixture, and theprecipitate was filtered off. To further purify the product, theprecipitate was dissolved in chloroform, and acetone was added tore-precipitate the product. The excess solvent was decanted off, and theprecipitate was dried using a high vacuum pump to yield a linear N-alkylN-methyl PEI polymer.

The polyethylenimine-based polymer typically has a number averagemolecular weight between 15,000 g/mol and 250,000 g/mol. Thepolyethylenimine-based polymer can have a number average molecularweight of 250,000 g/mol or less, for example, 230,000 g/mol or less,210,000 g/mol or less, 190,000 g/mol or less, or 170,000 g/mol or less.Alternatively, or in addition, the polyethylenimine-based polymer canhave a number average molecular weight of 15,000 g/mol or more, forexample, 30,000 g/mol or more, 60,000 g/mol or more, 90,000 g/mol ormore, 100,000 g/mol or more, 120,000 g/mol or more, or 150,000 g/mol ormore. Thus, the polyethylenimine-based polymer can have a number averagemolecular weight bounded by any two of the aforementioned endpoints. Forexample, the polyethylenimine-based polymer can have a number averagemolecular weight between 15,000 g/mol and 250,000 g/mol, between 15,000g/mol and 230,000 g/mol, between 15,000 g/mol and 210,000 g/mol, between15,000 g/mol and 190,000 g/mol, between 15,000 g/mol and 170,000 g/mol,between 30,000 g/mol and 170,000 g/mol, between 60,000 g/mol and 170,000g/mol, between 90,000 g/mol and 170,000 g/mol, between 120,000 g/mol and170,000 g/mol, or between 150,000 g/mol and 170,000 g/mol, e.g., about160,000 g/mol.

An aspect of the invention is an antimicrobial composition comprising(a) a polydiallyldialkylammonium salt (e.g., polydiallyldimethylammoniumhalide) is used in combination with a polyethylenimine-based polymer(e.g., a linear or branched polyethylenimine (PEI), preferably linearPEI), (b) at least one adhesion promoter, (c) optionally organic and/orinorganic particles that are photocatalytically active in visible light,(d) optionally at least one salt, and (e) a carrier, each of which isdescribed herein. The antimicrobial composition passes at least one oftests (i)-(v). The weight ratio of polydiallyldialkylammonium salt topolyethylenimine-based polymer is any suitable amount, but typicallyranges from 80/20 to 20/80 (e.g., 30/70, 33/67, 40/60, 45/55, 50/50,55/45, 60/40, 67/33, 70/30). In a particular example, the weight ratioof polyDADMAC:PEI is 50/50 or 33/67.

When the cationic polymer is a polyethylenimine-based polymer, thecomposition can further comprise an anionic polymer, such that thecationic polymer and anionic polymer combine to form a PEC. In someembodiments, the polyethylenimine-based polymer, such as non-chemicallymodified linear PEI, is used without an anionic polymer, such as apolyacrylic acid salt. In other embodiments in which a PEC is desired,the composition comprises both a polydiallyldialkylammonium salt (e.g.,polydiallyldimethylammonium halide) and PEI, such as a branched PEI. Twoapproaches are suggested for adding PEI to the system. One approach isto first complex the polydiallyldialkylammonium halide and anionicpolymer and then complex the PEI to the anionic polymer, followed by theblending of the two complexes. The second, and preferred, approach is tosimultaneously complex both cationic polymers with the anionic polymerin a one-pot synthesis.

It is known by those skilled in the art that non-enveloped viruses areresistant to ethanol, which is the alcohol most commonly used in handsanitizers and other disinfectants. The inventors discovered that acomposition of ethanol and non-chemically modified linear PEI iseffective at killing non-enveloped viruses and that the antimicrobialactivity can be further improved by adding an organic small moleculepolyacid, such as citric acid. Without wishing to be bound by anytheory, it is believed that protonated liner PEI binds the anionic formof the polyacid (e.g., citrate) to form a complex. Suitable organicpolyacids include a polycarboxylic acid comprising at least threecarboxylic acid groups (e.g., 3, 4, 5, and/or 6 carboxylic acid groups),such as an organic tribasic acid. Specific examples of a polycarboxylicacid include citric acid, isocitric acid, aconitic acid,propane-1,2,3-tricarboxylic acid, hemimelitic acid, trimellitic acid,trimesic acid, prehnitic acid, meallanophanic acid, pyromellitic aicd,benzenepentacarboxylic acid, mellitic acid,ethylenediamine-N,N′-dimalonic acid (EDDM), 2,2′-azanediyldisuccinicacid, 2,2′-oxydisuccinic acid (ODS), ethylenediaminedisuccinic acid(EDDS), diethylenetriaminepentaacetic acid (DTPA), ethylene diaminetetraacetic acid (EDTA),2,2′-((((1,2-dicarboxyethyl)azanediyebis(ethane-2,1-diyl))bis(oxy))disuccinicacid, and any combination thereof. Preferably the polyacid is citricacid. A PEI-citrate complex forms a stable colloidal when the ratio ofprotonated linear PEI to citric acid ranges from about 70:30 to 90:10(e.g., about 70:30, about 75:25, about 80:20, about 85/15, or about90:10). If more citric acid is desired in the complex, for example60:40, the colloidal may become unstable. However, the colloidal can bemade stable by filtering out the larger citrate complexes.

In another embodiment, the cationic polymer is chitosan. When thecationic polymer is chitosan, formation of a PEC is optional, i.e., ananionic polymer is optionally present in the composition. In someinstances, chitosan with 95% or less deacetylation and/or deacetylationwith quarternization (e.g., trimethylchitosan) results in a more solublehigh molecular weight chitosan. Thus, it is possible to create a lowsoluble film durable enough to not require the formation of a PEC.

The chitosan typically has a number average molecular weight between20,000 g/mol and 2,000,000 g/mol. The chitosan can have a number averagemolecular weight of 2,000,000 g/mol or less, for example, 1,750,000g/mol or less, 1,500,000 g/mol or less, or 1,250,000 g/mol or less.Alternatively, or in addition, the chitosan can have a number averagemolecular weight of 20,000 g/mol or more, for example, 50,000 g/mol ormore, 100,000 g/mol or more, 250,000 g/mol or more, 500,000 g/mol ormore, or 1,000,000 g/mol or more. Thus, the chitosan can have a numberaverage molecular weight bounded by any two of the aforementionedendpoints. For example, the chitosan can have a number average molecularweight between 20,000 g/mol and 2,000,000 g/mol, between 20,000 g/moland 1,750,000 g/mol, between 20,000 g/mol and 1,500,000 g/mol, between20,000 g/mol and 1,250,000 g/mol, between 20,000 g/mol and 1,000,000g/mol, between 50,000 g/mol and 2,000,000 g/mol, between 100,000 g/moland 2,000,000 g/mol, between 250,000 g/mol and 2,000,000 g/mol, between500,000 g/mol and 2,000,000 g/mol, or between 1,000,000 g/mol and2,000,000 g/mol.

When the antimicrobial composition optionally comprises at least oneanionic polymer, which forms a PEC with the cationic polymer, the PECscan offer two important advantages to the present invention: 1) theassembly of polymers using PECs eliminates the use of chemicalcross-linking agents, thereby reducing possible toxicity and otherundesirable effects of the reagents; and 2) the PECs formed between apoly acid and poly base are tolerant of pH variations in the dissolutionmedium.

The anionic polymer can be any suitable anionic polymer that is capableof forming a PEC with the cationic polymer, such as an anionic polymerselected from a polyacrylic acid salt, a polysulfate, a polysulfonate, apolycarboxylate, a polyoxometalate, a sulfonated or carboxylatedmetalloporphyrin, xanthan gum, alginate, or a lignin compound (e.g.,lignosulfonate, pectin, carrageenan, humate, fulvate, angico gum, gumKondagogu (Cochlospermum gossypium DC), sodium alkyl naphthalenesulfonate (e.g., MORWET™), poly-γ-glutamic acid, maleic starchhalf-ester, carboxymethyl cellulose, chondroitin sulphate, dextransulphate, and hyaluronic acid). The anionic polymer can be linear,branched, dendritic, graft, or present as a copolymer (e.g., blockcopolymer).

In preferred embodiments, the anionic polymer is a polyacrylic acid salt(PAAS). Specific examples of PAAS include polyacrylic acid alkali metalsalts (e.g., polyacrylic acid sodium salt) and polyacrylic acid ammoniumsalts. The polyacrylic acid salt has a number average molecular weightof at least 10,000 g/mol. For example, the polyacrylic acid salt canhave a number average molecular weight of 20,000 g/mol or more, e.g.,40,000 g/mol or more, 60,000 g/mol or more, 80,000 g/mol or more,100,000 g/mol or more, 120,000 g/mol or more, or 140,000 g/mol or more.

The size and internal structure of the PEC particles are regulated by,for example, the formation process, media and structural parameters,particular mixing order, mixing ratio, PEC concentration, pH, andmolecular weight. Controlling the size of the PEC particle is importantbecause the particle size affects 1) the overall stability of thePickering PEC; 2) the solvency of the film formed by the dispersion; and3) the adhesive strength of the film to a substrate. The solvency andadhesiveness of the film can be tuned by controlling the size of thefinal PEC colloidal. Some applications might require a less soluble andmore adhesive film. However, tuning these two attributes will always beconstrained by the stability issue. If too much anionic polymer is used,the PEC colloidal will become too large and precipitate out.

The size of the final PEC particle can be determined by the quantity ofanionic polyelectrolyte (n−) relative to the quantity of cationicpolyelectrolyte (n+). If the n−/n+ ratio is high, the PEC particle willgrow. However, when the cationic polymer is dosed into the anionicpolymer, there is a point of accelerated growth and then a fall-off insize. The preferred method of doping is to dose the anionic polymer intothe cationic polymer, even though this order of dosing cannot create thesmallest particles (assuming n−/n+ is below 0.8).

The size of the PEC particle is also influenced by the mixing order.When the anionic polymer is dosed into cationic polymer, the PECparticle becomes larger. Nevertheless, there are techniques to offsetthis undesirable growth. First, the size of the PEC emulsion particlescan be kept small by limiting the concentration of polyelectrolyte inthe forming solution; in other words, work with a very dilute solution.Given that the preferred mixing order has a detrimental effect onparticle size, the offsetting strategy put forth in the presentinvention is to work with dilute solutions, i.e., limit theconcentration of the polymers and then evaporate off the excess waterafter the formation of the PECs. In a specific embodiment of theinvention, the method prefers, but is not limited to, using a cationicpolymer (e.g., polydiallyldialkylammonium salt,acryloxyalkyltrialkylammonium salt, vinylphenalkyltrialkylammonium salt,acrylamidoalkyltrialkylammonium salt, PEI, and/or chitosan) at aconcentration of about 0.001 to 0.1 M (e.g., 0.005 M).

A second method of offsetting the tendency of the PEC to grow is tocontrol the pH of the cationic polymer mixture relative to the pH of theanionic polymer mixture. For example, a lower pH of the cationic polymer(pH ˜4) and high pH of the anionic polymer (pH ˜10) results in a smallerparticle size. Thus, the preferred, but not required, pH of the cationicpolymer (e.g., polydiallyldialkylammonium salt,acryloxyalkyltrialkylammonium salt, vinylphenalkyltrialkylammonium salt,acrylamidoalkyltrialkylammonium salt, PEI, and/or chitosan) solution forcreating the PEC is kept at ˜4 and the anionic pH is maintained at ˜10.The pH of the final PEC solution is ˜4.5 and after evaporation, the pHis adjusted to ˜7.4. It is believed that the lower pH of the cationicpolymer fluid contributes to a smaller particle size and thus helps tooffset the negative influence of dosing order and molecular weight,encouraging a larger particle size.

The antimicrobial composition should be kept at a pH near 7 in order topass the EPA cytotoxicity test. Even a slight dissolution of the filmduring the test can cause leaching, and a pH much above or below 7 willkill the mammalian cells used in the test and the film will fail. Also,a pH of 7 should help ensure that the anionic polymer is maintained inan ionized form. If necessary, the pH can be adjusted by the addition ofsuitable acids (e.g., hydrochloric acid, sulfuric acid, citric acid,etc.) or bases (e.g., sodium hydroxide, potassium hydroxide). It isrecommended that the final pH be adjusted subsequent to dispersing anyorganic and/or inorganic particles into the PEC.

With respect to the present invention, it is important that the PECsurface remains strongly positive. For example, if an excess quantity ofanionic polymer is added (i.e., if the (n−/n+) is too high) the PECparticle charge will become negative, which would destroy theeffectiveness of the antimicrobial composition, since it is believedthat the antimicrobial mode of action is related to the positivelycharged cationic polymer(s) attracting and piercing a negatively chargedmicrobial membrane. It is therefore important that the PEC particlecharge remains positive. For the purpose of the present invention, it isrecommended that (n−/n+) value does not exceed 0.3, and preferably isbelow 0.2.

In general, the ppm for the film thickness is determined by the amountof carrier (e.g., water) that is evaporated from the combined solution(e.g., PEC solution). When working with a very dilute concentration, theconsiderable excess carrier needs to be evaporated to arrive at adesirable ppm of solids in the film-forming composition.

It is an aspect of the present invention that the PECs are assembled insuch a way that the PECs have an average aggregate size in solution ofless than about 500 nm (e.g., less than 400 nm, less than 300 nm, lessthan 200 nm). In some embodiments, the aggregate size is less than about100 nm (e.g., less than 80 nm, less than 50 nm, less than 25 nm, lessthan 10 nm) in diameter. The particle size and molecular weights of theassociative PECs can be measured via static or dynamic light scattering.

The antimicrobial composition preferably also comprises at least oneadhesion promoter that allows the composition to adhere to the surfaceof a substrate to form a residual self-sanitizing film that cannot beimmediately washed away. In some embodiments, the residualself-sanitizing film is not covalently bound to the surface of thesubstrate. The adhesion promoter can, in some instances, be described asa coupling agent. The adhesion promoter typically is one or morecompounds with at least one functional group that has an attractiveforce to the surface of a desired substrate, the at least one cationicpolymer, or both. Suitable examples of an adhesion promoter include atitanate, carboxylated branched or linear PEI, a silane compound,cationic block copolymers, and other polymers that will create “sticky,”reactive groups, such as acyl or carboxylic acid, and carboxylic acidderivatives. Preferably, the adhesion promoter is a carboxylatedbranched PEI, as it does not detract from the cationic charge of thepolymers.

The titanate can be any suitable titanate that increases thecomposition's ability to adhere to a surface and/or enables thecomposition or a film formed from the composition to pass one or more oftests (i)-(v). Typically, the titanate is selected from an alkoxytitanate, a neoalkoxytitanate, an oxyacetate chelated titanate, anethylene chelated titanate, a pyrophosphate titanate, and combinationsthereof.

In preferred embodiments, the titanate is selected from titanium IV2,2(bis 2-propenolatomethyl)butanolato, tris neodecanoato-O, titanium IV2,2(bis 2-propenolatomethyl)butanolato, tris(dodecyl)benzenesulfonato-O,titanium IV 2,2(bis 2-propenolatomethyl)butanolato,tris(dioctyl)phosphato-O, titanium IV 2,2(bis2-propenolatomethyl)butanolato, tris(dioctyl)pyrophosphato-O, titaniumIV 2,2(bis 2-propenolatomethyl)butanolato,tris(2-ethylenediamino)ethylato, titanium IV 2,2(bis2-propenolatomethyl)butanolato, tris(3-amino)phenylato, titanium IV2,2(bis 2-propenolatomethyl)butanolato, tris(6-hydroxy)hexanoato-O, orany combination thereof. Typically, the titantate is titanium IV 2,2(bis2-propenolatomethyl)butanolato, tris neodecanoato-O.

The antimicrobial composition can comprise any suitable amount oftitanate to form a residual self-sanitizing film. The antimicrobialcomposition can comprise, for example, titanate in an amount of 0.1%weight based on monomers (“wbm”) of the cationic polymer, or more, forexample, 0.2% wbm or more, 0.3% wbm or more, 0.4% wbm or more, or 0.5%wbm or more. Alternatively, or in addition, the antimicrobialcomposition can comprise titanate in an amount of 6% wbm of the cationicpolymer, or less, for example, 5% wbm or less, 4% wbm or less, 3% wbm orless, 2% wbm or less, 1% wbm or less, 0.9% wbm or less, 0.8% wbm orless, or 0.7% wbm or less. Thus, the antimicrobial composition cancomprise titanate in an amount bounded by any two of the aforementionedendpoints. For example, the antimicrobial composition can comprisetitanate in an amount between 0.1% wbm to 6% wbm of the cationicmonomers, for example, between 0.2% wbm to 6% wbm, between 0.3% wbm to6% wbm, between 0.4% wbm to 6% wbm, between 0.5% wbm to 6% wbm, between0.5% wbm to 5% wbm, between 0.5% wbm to 4% wbm, between 0.5% wbm to 3%wbm, between 0.5% wbm to 2% wbm, between 0.5% wbm to 1% wbm, between0.5% wbm to 0.9% wbm, between 0.5% wbm to 0.8% wbm, or between 0.5% wbmto 0.7% wbm, e.g., 0.6% wbm.

The adhesion promoter can be carboxylated PEI (PEI-COOH) that is eitherbranched, linear, or a mixture of branched and linear. The PEI-COOH canbe purchased commercially or prepared from PEI. For example, bromoaceticacid in water can be added to PEI in water. The resulting mixture isthen stirred and then filtered to isolate the polymer and removeunreacted acid. The PEI-COOH can have any suitable molecular weight buttypically has a number average molecular weight between 15,000 g/mol and250,000 g/mol. The PEI-COOH can be used in a suitable amount thatusually ranges from 0.001% to 3% by weight, including ranges with endpoints at 0.01%, 0.1%, 0.5%, 1%, 1.5%, 2%, and/or 2.5%. A preferredamount ranges from 0.001% to 0.01%, such as 0.001% by weight.

A silane compound, such as a silane coupling agent, can be used as theadhesion promoter. In general, a silane coupling agent has functionalgroups at both terminal ends that allow an organic group, such as thecationic polymer, to bond to an inorganic group, such as a substrate.The silane compound can have the formula R—(CH₂)_(n)—Si—X₃, in which Ris an organofunctional group (e.g., optionally substituted linear orbranched C₁-C₂₀ alkyl, optionally substituted aryl, such as phenyl ornaphthyl, amino, such as —NH(CH₂)₃NH₂, epoxy, or methacryloxy), n is aninteger from 0 to 6, and X is a hydrolyzable group (e.g., alkoxy,acyloxy, halo, or amino). Suitable examples include a trialkoxysilaneand a monoalkoxysilane, in which the alkoxy is C₁-C₆ alkoxy (e.g.,methoxy, ethoxy, propoxy, isopropoxy, butoxy, or a combination thereof),a dipodal (branched) silane with two alkoxy-silane branches, a cyclicazasilane, a vinyl silane, an acryloxy silane, an epoxysilane, and anaminosilane, or any combination thereof. Specific examples of silanecompounds include methyltrimethoxysilane, methyltriethoxysilane,isobutyltrimethoxysilane, n-octytriethoxysilane, phenyltrimethoxysilane,vinyltrichlorosilane, vinyltris(β-methoxyethoxy)silane,vinyltriethoxysilane, vinyltrimethoxysilane,3-metacryloxypropyl-trimethoxysilane,β-(3,4-epoxycyclohexyl)-ethyltrimethoxysilane,γ-glycidoxypropyl-trimethoxysilane,γ-glycidopropyl-methylidiethoxysilane,N-β(aminoethyl)-γ-aminopropyl-trimethoxysilane,N-β(aminoethyl)-γ-aminopropyl-methyldimethoxysilane,3-aminopropyl-triethoxysilane, andN-phenyl-γ-aminopropyl-trimethoxysilane, or a combination thereof.

The adhesion promoter can also be a cationic block copolymer, such ahigh molecular weight polyethylene-based copolymer with basic or acidicadhesive groups, such as an amino and/or hydroxy. Commercial products ofthis type include BYK™ 4500, BYK™ 4510, BYK™ 4509, BYK™ 4512, and BYK™4513, which are available from BYK Chemie GmbH (Wesel, Germany).Suitable amounts of the block copolymer range from 0.001% to 5% byweight, including ranges with end points at 0.01%, 0.1%, 0.5%, 1%, 1.5%,2%, 2.5%, 3%, 3.5%, 4%, or 5%. A preferred amount ranges from 0.5% to2%, such as 1% by weight.

The adhesion promoter can also be a polymer that either naturally has orhas been modified to have “sticky,” reactive groups, such as an acylgroup, a carboxylic acid, a carboxylic acid derivative, asulfur-containing moiety (e.g., thio), an amino group, hydroxyl, and/ora halo-containing group. The polymer itself is any suitable moiety,preferably without a charge, such as polyethylene, polypropylene,poly(ethylene-vinylacetate), polyester, polyurethane, polyamide,polyvinyl acetate, polyvinyl alcohol, polyvinyl butyral, polyvinylchloride, polyvinyl ether, or a combination thereof. Suitable amounts ofthe polymer range from 0.001% to 3% by weight, including ranges with endpoints at 0.01%, 0.1%, 0.5%, 1%, 1.5%, 2%, and/or 2.5%. A preferredamount ranges from 0.5% to 2%, such as 1% by weight.

In some embodiments, the antimicrobial composition comprises organicand/or inorganic particles that are photocatalytically active in visiblelight and can be any suitable organic-based (e.g., graphene or graphiticcarbon nitride (g-C₃N₄)) and/or inorganic-based particles that arephotocatalytically active in visible light (e.g., between 390 to 700nm). Organic and/or inorganic particles that are photocatalyticallyactive generate reactive oxygen species that are able to destroypathogenic microbes (e.g., kills C. difficile, bacteria and/or viruses,including avian flu and SARS) that augments the disinfectant property ofthe composition. Generally, the organic and/or inorganic particles thatare photocatalytically active in visible light are selected fromgraphene, g-C₃N₄, a transition metal oxide, a transition metal sulfide,a transition metal selenide, a dye sensitizer, a conjugated polymer, anoble metal, or a mixture thereof. A mixture of particles means that twoor more different kinds of particles are present in the antimicrobialcomposition, whereas in a multijunction composite, various components ofthe composite are tightly coupled to assure electron transfer, andminimize recombining of holes.

As used herein, the term “particle” includes sphere-like particles(e.g., spheres) and other shapes, such as platelets, rods, cubes, andflakes or combinations of various shapes and morphologies.

Graphene is an allotrope of carbon, in which carbon atoms are bonded toone another in sheet form that is one atom thick. The graphene canoptionally be functionalized with an oxygen- and/or nitrogen-containinggroup. An analog of graphite is graphitic carbon nitride (g-C₃N₄), whichis photocatalytic.

The transition metal oxide, sulfide, and selenide can be any suitablecompound comprising at least one metal atom and at least one anion ofoxygen, sulfur, or selenium that has an oxidation state of −2. In someaspects, the transition metal oxide is selected from the groupconsisting of silicon dioxide (including fumed silica, amorphous silica,precipitated silica, hydrophilic silica, and hydrophobic silica),titanium dioxide, zinc oxide, iron oxide, aluminum oxide, cerium oxide,zirconium oxide, and a combination thereof; the transition metal sulfideis selected from cadmium sulfide, molybdenum disulfide, tungstensulfide, silver sulfide, zinc sulfide, selenium sulfide, iron disulfide,nickel sulfide, ruthenium sulfide, cobalt sulfide, and a combinationthereof; and/or the transition metal selenide is selected from cadmiumselenide, copper selenide, copper geranium selenide, copper indiumgallium selenide, copper titanium selenide, indium selenide, manganesediselenide, titanium selenide, tungsten diselenide, silver selenide,disilver selenide, digold triselenide, zinc sulfide, iron selenide,nickel selenide, ruthenium selenide, cobalt selenide, and a combinationthereof.

Tungsten doping, as well as other metallic doping, has been demonstratedto inhibit charge recombination and improve photocatalytic activity ofphotocatalysts (Rozenberg et al., Prog Polym Sci, 2008, 33: 40-112). Incertain embodiments, the transition metal oxide/sulfide/selenideparticle can be doped with a suitable metal, such as tungsten, nitrogen,or a combination of tungsten and nitrogen.

In one embodiment, the transition metal oxide is titanium dioxide(TiO₂). The TiO₂ particles can be obtained from any suitable mineralform of TiO₂. For example, the TiO₂ particles can maintain an anatasecrystalline structure, a brookite crystalline structure, or a rutilecrystalline structure. In preferred embodiments, the TiO₂ maintains ananatase crystalline structure.

The TiO₂ particles can be any suitable structure type. Typically, theTiO₂ particles are TiO₂ nanoparticles (“NP”). The TiO₂ nanoparticles canbe synthesized by any suitable process. For example, the TiO₂nanoparticles can be liquid-synthesized or gas-synthesized. In preferredembodiments, the TiO₂ nanoparticles are liquid-synthesized, since aliquid synthesis tends to create soft agglomerates, which make it easierto disperse the TiO₂. An illustrative TiO₂ nanoparticle is a 25 nm,anatase, liquid-phase synthesized TiO₂ nanoparticle doped with tungsten,which can be purchased from Nanostructured & Amorphous Materials, Inc.(Houston, Tex.).

In a specific example of functionalizing TiO₂ particles, W-doped, liquidsynthesized TiO₂ (20 nm) is calcined with urea at 400° C. for 1 hour,which produces a poly(amino-tri-s-triazine) polymer covalently attachedto the TiO₂ particles. Next, the WIN-doped TiO₂ particles are milledalong with powdered urea. The hard material created from calcination isground into powder so that it can be placed, along with urea, into aplanetary ball mill. The milling spec is to mill at 300 rpm for 30minutes with 10% urea and balls weighing 10 times the weight of theTiO₂. After 30 minutes, the milling drum is three-quarters filled with200 ml of H₂O and milled an additional 5 minutes to capture and dispersethe TiO₂ nanoparticles. The contents are then put into a beaker andmixed under 150W UV light for 1 hour. The highly dispersed nanopowder isthen available to be added to the antimicrobial composition. It isimportant to note that the nanopowder is highly dispersed in water,which keeps it in a non-agglomerated nano state. As such, thefunctionalization process described herein enables the TiO₂ to bedispersible in water, wherein TiO₂ is typically only dispersible inalcohol. Creating such a stable nano-dispersion without a surfactantmeans that when the particles are dispersed into a cationic polymersolution or the PEC, the particles will not be contaminated with asurfactant that could possibly dampen their ability to respond tovisible light.

The high-energy milling of TiO₂ particles achieves two things: 1) itde-agglomerates the powder to create nanoparticles; and 2) the ureadouble dopes the particles with nitrogen, and in particular, any newexposed particle facets are doped as the high-energy milling breaks upagglomerates and aggregates. Essentially, it is believed that themilling forces nitrogen into the pores and covers facets that were notpreviously exposed during the calcination process. Following calcinationwith urea and milling with urea, the TiO₂ nanoparticles are irradiatedwith a 150 watt UV light. Without wishing to be bound by any theory, itis believed that UV irradiation improves the visible lightresponsiveness of TiO₂ nanoparticles because of the introduction ofhydroxyl groups on to the surface of the TiO₂ nanoparticles. This is oneexplanation for why the particles are easily dispersed in water. Theability of the functionalized nanoparticles to degrade methylene bluewas tested, and it was observed that the sum of all fourfunctionalization steps significantly degraded the dye within 90minutes.

Lastly following calcination, milling, washing, and light irradiation,the particles can be dye-sensitized. The theory and practice of usingdye to enhance the visible-light sensitivity of a transition metal oxideparticle (e.g., TiO₂) is central to “dye-sensitized solar cell” (DSSC)technology. DSSCs have been attracting considerable attention in recentyears owing to their comparatively low cost and high efficiency. A DSSCis essentially a photo-electrochemical system, in which light harvestingis accomplished by dye molecules that are adsorbed on the surface of theoxide nanostructures that form the photo-electrode film. Surfacesensitization of a wide band gap semiconductor photo-catalyst, such asTiO₂, via chemisorbed or physisorbed dyes can increase the efficiency ofthe excitation process and expand the wavelength range of excitation forthe transition metal oxide particle (e.g., TiO₂). This occurs throughexcitation of the sensitizer that can inject either a hole or, morecommonly, an electron into the particle. Highly efficient chargeinjection is observed when a monolayer of a dye is dispersed on aphotocatalyst with a high surface area. This sensitization increases therange of the wavelength response of the photocatalyst, which isimportant for it to operate under natural sunlight. The electroninjection and back electron-transfer rates from the dye to thetransition metal oxide particle (e.g., TiO₂) depend on the nature of thedye molecule, the properties of the transition metal oxide particle(e.g., TiO₂), and the interactions between the dye and the transitionmetal oxide particle. The dye is any suitable compound, such asfluorescein, fluorescein isothiocyanate, a cyanine, a merocyanine, ahemicyanine, a perylene, a xanthene, a porphyrin (e.g.,tetraphenylporphyrin), a phthalocyanine (e.g., copper phthalocyanine), apolyene, a polythiophene, a coumarin (e.g., NKX-2677, NKX-2587,NKX-2697, NKX-2753, NKX-2586, or NKX-2311), and a ruthenium-based dye(e.g., (Bu₄N)₂[Ru(dcbpyH)₂(NCS)₂] (N719), (Bu₄N)₂[Ru(dcbpy)₂(NCS)₂],cis-di(thiocyanato)bis(2,2′-bipyridyl-4,4′-dicarboxylate) ruthenium(II)(N3),tri(thiocyanato)-2,2′,2″-terpyridyl-4,4′,4″-tricarboxylate)ruthenium(II)(black dye), K8, K9, K19, and Z907). In a specific embodiment of theinvention, N719 dye is applied by mixing calcinated/milled/UV lightfunctionalized transition metal oxide particles (e.g., TiO₂) for 1 hourin the dark with a 0.5 mM mixture of N719 dye in ethanol. Other dyes canalso be used. The functionalized particles are decanted, centrifuged,and added back to water.

In any of the embodiments described herein, the TiO₂ particles are dopedwith tungsten and nitrogen and are hydrolyzed under ultraviolet (UV)light. The resulting particles are visible light-responsive TiO₂particles that are effective as an antimicrobial agent, particularlywhen such particles are embedded in a film formed from an antimicrobialcomposition of the present invention. Accordingly, the inventionprovides a method of killing microbes on a surface (e.g., disinfecting asurface, providing a residual self-sanitizing film, or both) comprisingapplying to the surface an antimicrobial composition comprising (i)visible light-responsive TiO₂ particles that are doped with tungsten andnitrogen, (ii) at least one adhesion promoter (e.g., a titanate, acarboxylated branched PEI), and (iii) a carrier. The adhesion promoteris as described herein, and the carrier can be, e.g., water, alcohol, ora combination of water and alcohol, as described herein.

Without wishing to be bound by any particular theory, the electronicstructure of TiO₂ is characterized by a filled valence band and an emptyconduction band. The band gap energy is excited and an electron ispromoted from the valence band to the conduction band and anelectron-hole pair is generated. This electron hole reacts with water togenerate active oxygen such as hydroxyl radicals, sometimes referred toas reactive oxygen species (ROS). The positive hole of TiO₂ breaks thewater molecule apart to form hydrogen gas and hydroxyl radicals. Thenegative electron reacts with oxygen molecules to form a super-oxideanion (O₂ ⁻). Super oxide anions further react with water molecules togenerate hydroxyl radical peroxide (•OOH) and hydrogen peroxide (H₂O₂).Each •OH, O₂ ⁻, •OOH, and H₂O₂ can react with pathogenic microbes anddestroy their cell structure.

In addition, the electron holes themselves can directly react with themicrobial cell wall, cell membrane, and cell components. In microzymesand bacilli, intracellular coenzyme A (CoA) is oxidized by TiO₂ suchthat the CoA dimer loses its activity, which causes the respiration ofthe cell to stop and finally results in microbial death. During thisprocess, the electron shift between the killed cell and TiO₂ is passedthrough CoA. Therefore, the content of CoA decreases and the CoA dimerincreases.

The dye sensitizer is any suitable compound, such as fluorescein,fluorescein isothiocyanate, a cyanine, a merocyanine, a hemicyanine, aperylene, a xanthene, a porphyrin (e.g., tetraphenylporphyrin), aphthalocyanine (e.g., copper phthalocyanine), a polyene, apolythiophene, a coumarin (e.g., NKX-2677, NKX-2587, NKX-2697, NKX-2753,NKX-2586, or NKX-2311), and a ruthenium-based dye (e.g.,(Bu₄N)₂[Ru(dcbpyH)₂(NCS)₂](N719), (Bu₄N)₂[Ru(dcbpy)₂(NCS)₂],cis-di(thiocyanato)bis(2,2′-bipyridyl-4,4′-dicarboxylate) ruthenium(II)(N3),tri(thiocyanato)-2,2′,2″-terpyridyl-4,4′,4″-tricarboxylate)ruthenium(II)(black dye), K8, K9, K19, and Z907).

The organic and/or inorganic photocatalytic particles can include aconjugated polymer that conducts. A suitable conjugated polymer includespolypyrrole (Ppy), poly(3-hexylthiophene) (P3HT), polycarbazole,polyindole, polyazepine, polyaniline, polyfluorene, polyphenylene,polypyrene, polyazulene, polynaphthalene, polythiophene (Ptp),poly(3,4-ethylenedioxythiophene), poly(p-phenylene sulfide),polyacetylene, poly(p-phenylene vinylene), and any combination thereof.The conjugated polymer can be incorporated into a nanocomposite that isspecifically designed to react with ordinary room lighting to createreactive oxygen species (ROS). The ROS destroy difficult-to-killspore-like microbes, such as C. difficile and fungi. The ROS alsooxidize microbial debris, thus performing an ongoing cleaning function.In a particular embodiment, the photocatalytic nanocomposite iscomprised of a multi-junction composite comprising: WTiO₂/CNheterojunction/Ppy, in which WTiO₂ is tungsten-doped TiO₂ nanoparticles,as described herein, CN is graphitic carbon nitride (g-C₃N₄), and Ppy ispolypyrrole.

Historically, most photocatalytic materials have been designed aroundvarious metal sulfides and metal oxides rather than polymers. Titaniumdioxide (TiO₂) has been the metal oxide of choice because it is readilyavailable, inexpensive, stable, non-toxic, and highly reactive in theultraviolet (UV) light spectrum. The inventors wanted to go beyond theuse of TiO₂ because the goal was to create a photocatalytic materialthat would be reactive in ordinary room lighting. As TiO₂ has a wideband gap (3-3.2 eV), it only absorbs light in the UV spectrum, none ofwhich is present in indoor room lighting. Thus, the present inventionprovides a photocatalytic nanocomposite that is based upon polymers,i.e., with no or very limited use of metals. Such nanocomposite islikely to be less toxic for humans and the environment and has noanionic charge to off-set the cationic charge when incorporated in acationic polymer-containing residual self-sanitizing film, as describedherein. In a nanocomposite based more on polymers, WTiO₂ in the WTiO₂/CNheterojunction/Ppy multi-junction composite is replaced with anacid-modified or protonated graphitic carbon nitride (g-C₃N₄). Theprotonation of CN gives the CN a band gap with valance and conductivebands that are quite close to TiO₂. The inventors have developed severaltechniques to create the protonated CN, which is termed acidified carbonnitrate (ACN), and then strongly couple the protonated CN to theheterogeneous CN and a conjugated polymer (such as polypyrrole (Ppy),poly(3-hexylthiophene) (P3HT), polythiophene (Ptp), and the like),thereby resulting in a photocatalytic composite specifically engineeredto capture low level indoor light. This method is explained in detail inU.S. Provisional Patent Application 62/367,981 and the inventors'concurrently filed provisional patent application, the entire contentsof which are incorporated herein by reference.

The benefits of this new, polymer-based, photo-reactive material includeone or more of the following: (i) maximum light harvesting withmulti-junction band slicing, (ii) maximum photon utilization by usingmaterials with appropriate band edges, sequencing the assembly and tightcoupling to foster rapid electron transport, and minimizingelectron-hole recombination, (iii) assembly using a low-cost, easilyscalable manufacturing process that does not use toxic chemicals orgenerate waste, and that creates a Nano, mesoporous material with anexceedingly high surface area that creates a mixed morphology stabledispersion of micro, nano, and crystalline particles and platelets thatmaximizes the optical path of the incident light and preserves all“unseen” nano/crystalline particles and platelets.

The organic and/or inorganic photocatalytic particles can include anoble metal, such as ruthenium, rhodium, palladium, silver, osmium,iridium, platinum, gold, or a mixture thereof. In some embodiments, thenoble metal is platinum.

The average diameter of the organic and/or inorganic particles that arephotocatalytically active in visible light is not particularly limitedand can range from 5 nm to 1,000 nm. The organic and/or inorganicparticles can have an average diameter of 1,000 nm or less, for example,750 nm or less, 500 nm or less, 250 nm or less, or 100 nm or less.Alternatively, or in addition, the organic and/or inorganic particlescan have an average diameter of 5 nm or more, for example, 10 nm ormore, or 15 nm or more. Thus, the organic and/or inorganicphotocatalytic particles can have an average diameter bounded by any twoof the aforementioned endpoints. For example, the organic and/orinorganic photocatalytic particles can have an average diameter of 5 nmto 1,000 nm, 5 nm to 750 nm, 5 nm to 500 nm, 5 nm to 250 nm, 5 nm to 100nm, 10 nm to 100 nm, or 15 nm to 100 nm.

The antimicrobial composition can comprise any suitable amount oforganic and/or inorganic photocatalytic particles to form a residualself-sanitizing film. The antimicrobial composition can comprise organicand/or inorganic photocatalytic particles in an amount of 1% weightbased on monomers (“wbm”) of the cationic monomers, or more, forexample, 1.5% wbm or more, 2% wbm or more, 2.5% wbm or more, 3% wbm ormore, 4% wbm or more, or 5% wbm or more. Alternatively, or in addition,the antimicrobial composition can comprise organic and/or inorganicparticles in an amount of 20% wbm or less of the cationic polymer, orless, for example, 18%% wbm or less, 15% wbm or less, 12% wbm or less,10% wbm, 9% wbm or less, 8% wbm or less, 7% wbm or less, 6% wbm or less,or 5% wbm or less. Thus, the antimicrobial composition can compriseorganic and/or inorganic particles in an amount bounded by any two ofthe aforementioned endpoints. For example, the antimicrobial compositioncan comprise organic and/or inorganic particles in an amount between 1%wbm to 20% wbm of the cationic monomers, for example, between 1% wbm to15% wbm, between 1% wbm to 10% wbm, between 1% wbm to 7% wbm, between 1%wbm to 6% wbm, between 1% wbm to 5% wbm, between 4% wbm to 20% wbm,between 5% wbm to 15% wbm, between 4% wbm to 8% wbm, or between 5% wbmto 8% wbm.

In an aspect of the invention, an antimicrobial composition comprises atleast one organic and/or inorganic particle that is photocatalyticallyactive in visible light, at least one adhesion promoter, and a carrier.The organic and inorganic photocatalytic particles, adhesion promoter,and carrier are described herein. A film formed from an antimicrobialcomposition comprising a photocatalytic particle kills microbes underthe conditions of the modified protocol for JIS Z 2801 (2006 version,which was updated in 2010). For example, an antimicrobial compositioncomprising at least one organic and/or inorganic particle that isphotocatalytically active in visible light kills at least 90% (e.g., atleast 92%, at least 94%, at least 96%, at least 97%, at least 98%, atleast 99%) of a log 4 population of Clostridium difficile bacteria,fungus, or yeast within 24 hours of contact. As such, the compositioncan be used in accordance with any of the methods described herein tokill microbes on a surface.

In some embodiments, the antimicrobial composition comprises a salt,which can affect the ability of the cationic polymer to adsorb to thesurface of a substrate and create a film. Without wishing to be bound byany theory, it is believed that high salt concentrations causeconditions similar to the interactions experienced by a polymer in afavorable solvent. Polyelectrolytes, while charged, are still mainlynon-polar with carbon backbones. While the charges on the polymerbackbone exert an electrostatic force that drives the polymer into amore open and loose conformation, if the surrounding solution has a highconcentration of salt, then the charge repulsion will be screened. Oncethis charge is screened, the polyelectrolyte will act as any othernon-polar polymer would in a high ionic strength solution and begin tominimize interactions with the solvent, which can lead to a much moreclumped and dense polymer deposited onto the surface and improvedadsorption or adhesion.

The salt is any inorganic salt, such as any salt containing a cation ofa Group I metal (lithium, sodium, potassium, rubidium, or cesium), aGroup II metal (beryllium, magnesium, calcium, strontium, or barium),ammonium, or aluminum. The counter anion can be a halide, carbonate,bicarbonate, sulfate, thiosulfate, phosphate, nitrate, nitrite, acetate,bromate, chlorate, iodate, etc. Specific examples of salt includelithium bromide, lithium chloride, lithium iodate, lithium iodide,lithium hydroxide, lithium sulfate, lithium phosphate, sodium bromide,sodium chloride, sodium acetate, sodium bicarbonate, sodium bisulfate,sodium bromate, sodium chlorate, sodium hydrosulfide, sodium hydroxide,sodium hypophosphite, sodium iodate, sodium iodide, potassium acetate,potassium bicarbonate, potassium bromate, potassium bromide, potassiumchloride, potassium carbonate, potassium chlorate, potassium hydroxide,potassium iodide, potassium phosphate, potassium thiosulfate, rubidiumbromide, rubidium chloride, rubidium fluoride, rubidium iodide, rubidiumnitrate, rubidium sulfate, cesium bromide, cesium chloride, cesiumcarbonate, cesium nitrate, beryllium nitrate, beryllium sulfate,magnesium acetate, magnesium bromide, magnesium chloride, magnesiumiodate, magnesium iodide, magnesium nitrate, magnesium phosphate,magnesium sulfate, calcium acetate, calcium bromide, calcium chloride,calcium iodide, calcium iodate, calcium nitrite, calcium nitrate,calcium phosphate, calcium sulfate, strontium bromide, strontiumchloride, strontium hydrogen phosphate, strontium iodide, strontiumnitrate, strontium sulfate, barium acetate, barium bromide, bariumchloride, barium iodide, barium nitrate, barium phosphate, bariumsulfate, barium thiosulfate, ammonium acetate, ammonium bicarbonate,ammonium bromide, ammonium chloride, ammonium nitrate, aluminumchloride, aluminum phosphate, and any combination thereof. In someembodiments, the salt is a Group I-halide salt, such as sodium chlorideor potassium chloride.

The antimicrobial composition can comprise any suitable amount of salt,such as 0.01 M to 0.1 M, including any combination of endpoints at,e.g., 0.01 M, 0.02 M, 0.03 M. 0.04 M, 0.05 M, 0.06 M, 0.07 M, 0.08 M,0.09 M, and 0.1 M. In a specific example, the antimicrobial compositioncomprises 0.01 M to 0.05 M salt.

If desired, the cationic polymer can be blended with one or morenon-electrolyte (nonionic) polymers. A suitable non-electrolyte(nonionic) polymer preferably is water soluble and includes, forexample, a polyacrylamide, a polyamine, a polyamidoamine, polyethyleneglycol, polyvinyl pyrrolidone, polyvinyl alcohol, and a polyacrylate(e.g., poly(methyl)methacrylate), or any combination thereof.

The antimicrobial composition comprises a carrier. The carrier can beany suitable carrier that evaporates once the composition is applied toa desired surface. In general, the carrier is selected from an alcohol,water, or a combination thereof. In some embodiments, the carriercomprises a combination of water and alcohol. A suitable alcoholincludes methanol, ethanol, n-propanol, iso-propanol, n-butanol,sec-butanol, and t-butanol, or a combination thereof. In preferredembodiments, the carrier comprises ethanol (e.g., the carrier is acombination of ethanol and water). When a combination of alcohol andwater is used as the carrier, the ratio of alcohol:water preferablyranges from 10:90 to 99:1 (e.g., 10:90, 20:80, 30:70, 40:60, 50:50,60:40, 70:30, 80:20, 90:10, 95:5, and 99:1). In certain embodiments, thealcohol:water ratio ranges from 70:30 to 80:20.

In general, the antimicrobial composition does not comprise a germicidalsmall molecule compound (i.e., non-polymer) or antimicrobial metal,including those conventional germicidal agents that are EPA-registered,because such components have a material effect on the composition.EPA-approved germicidal agents that can be excluded from the compositioninclude, for example, glutaral, halazone, hexachlorophene,nitrofurazone, nitromersol, povidone-iodine, thimerosol, C₁-C₅-parabens,hypochlorite salts, clofucarban, clorophen, poloxamer iodine, phenolics,mafenide acetate, aminacrine hydrochloride, quaternary ammonium salts,oxychlorosene, metabromsalan, merbromin, dibromsalan, glyceryl laurate,sodium and/or zinc pyrithione, (dodecyl)(diethylenediamine)glycine,(dodecyl)(aminopropyl)glycine, a phenolic compound, (e.g., m-cresol,o-cresol, p-cresol, o-phenyl-phenol, 4-chloro-m-cresol, chloroxylenol,6-n-amyl-m-cresol, resorcinol, resorcinol monoacetate,p-tert-butylphenol and o-benzyl-p-chlorophenol), alkalineglutaraldehyde, and a quaternary ammonium salt (e.g., N-(higher)C₁₀-C₂₄-alkyl-N-benzyl-quaternary ammonium salts that comprisewater-solubilizing anions such as halide, e.g., chloride, bromide andiodide; sulfate, and methosulfate, and the heterocyclicimides such asthe imidazolinium salts). Suitable quaternary ammonium compounds aredescribed in U.S. Pat. No. 8,067,403 and include: benzalkonium chlorides(e.g., benzalkonium chloride), substituted benzalkonium chlorides (e.g.,alkyl dimethyl benzyl ammonium chloride), dual quaternary ammoniumcompounds (e.g., contain an equal mixture of alkyldimethyl benzylammonium chloride and alkyl dimethyl ethylbenzyl ammonium chloride),twin or dual chain quaternary ammonium compounds, such as dialkylmethylamines (e.g., didecyl dimethyl ammonium chloride or dioctyldimethylammonium chloride), and mixtures of fourth generation quaternaryammonium compounds with second-generation quaternary ammonium compounds(e.g., didecyl dimethyl ammonium chloride with alkyl dimethyl benzylammonium chloride). In an embodiment, the germicide is at least onemember selected from the group consisting of sodium hypochlorite,chloride, chlorine dioxide, sodium chloride, potassium persulfate,potassium permanganate, silver nitrate, chlordexidine, hexachlorophene,hydrogen peroxide, acetic acid, peracetic acid, betadine, povidoneiodine, formaldehyde, glutaraldehyde, benzalkonium chloride, triclosan,boric acid, phenol, cresylic acid, thymol, and polyhexamethylbiguanide.

However, if desired, one or more additional chemical germicidal agents,such as those agents described above, can be added to any of theforegoing embodiments of the antimicrobial composition. This optionprovides an additional chemical killing mechanism to further enhance theantimicrobial activity of the antimicrobial composition. When one ormore germicidal agents are incorporated into the antimicrobialcomposition, the agents are entrapped in the antimicrobial residualself-sanitizing film and are incrementally released when the coatedsurface comes into contact with moisture. When the film is exposed tolarger amounts of water, such as when the surface is moistened bywiping, food residues, or dishwater, this can lead to the release ofincreased amounts of the germicide. Thus, it is important that whatevergermicidal agents are used, the agents must not be toxic to humans, norshould they make the film tacky, hazy or in any way detract from theappearance of the surfaces to which they are applied. The germicidalagents are typically added in lower concentrations. Accordingly, suchadditives preferably comprise between 0.001% and 5% weight based onmonomers (“wbm”) of the cationic polymer.

In certain embodiments, the antimicrobial composition consistsessentially of or consists of a polydiallydimethylammonium halide, apolyethylenimine-based polymer, an anionic polymer, at least oneadhesion promoter (e.g., a titanate, a carboxylated branched PEI),optionally organic and/or inorganic particles that arephotocatalytically active in visible light, and a carrier, eachcomponent of which is described herein. In some aspects of thisembodiment, the organic and/or inorganic particles that arephotocatalytically active in visible light are present in thecomposition. In certain embodiments, the antimicrobial compositionconsists essentially of or consists of a polydiallydimethylammoniumhalide, a polyethylenimine-based polymer, at least one adhesionpromoter, optionally an anionic polymer, optionally organic and/orinorganic particles that are photocatalytically active in visible light,and a carrier, each component of which is described herein. In certainembodiments, the antimicrobial composition consists essentially of orconsists of a polydiallydimethylammonium halide, at least one adhesionpromoter (e.g., a titanate, a carboxylated branched PEI), organic and/orinorganic particles that are photocatalytically active in visible light,and a carrier, each component of which is described herein.

Another aspect of the invention is an antimicrobial compositioncomprising a polyethylenimine-based polymer, optionally a secondcationic polymer selected from a polydiallyldialkylammonium salt, apoly(acrylamide-co-diallyldialkylammonium halide), chitosan, or acombination thereof, optionally a polyacid, optionally at least oneadhesion promoter, and a carrier. The polyethylenimine-based polymertypically is linear or branched polyethylenimine (PEI), as describedherein, but preferably is linear PEI that is not chemically orstructurally modified.

In some instances, the second cationic polymer is absent. In otherinstances, the second cationic polymer is a polydiallyldialkylammoniumsalt that is a polydiallyldimethylammonium halide (e.g.,polydiallyldimethylammonium chloride and/or polydiallyldimethylammoniumfluoride). In an embodiment, the second cationic polymer is apoly(acrylamide-co-diallyldialkylammonium halide), such aspoly(acrylamide-co-diallyldimethylammonium chloride). In otherembodiments, the second cationic polymer is chitosan.

The polyacid and at least one adhesion promoter are as described herein.

The carrier used in the composition is any suitable carrier, asdescribed herein (e.g., water, propanol, iso-propanol, and/or ethanol).Typically, the composition will comprise a blend of 20% to 80% by volumeof various blends of alcohol with the balance made up with water. Inorder to increase the virucidal action, between 3% and 10% of variousblends of diols, preferably those with a chain length of from 3 to 5carbon atoms, such as a propanediol (1,2-propanediol and1,3-propanediol) or butanediols (1,3-butanediol), can be added to thecomposition. Preferably the diol is 1,2-propanediol and/or the alcoholis ethanol.

A proton donor can be added to the composition in a suitable amount(e.g., about 0.015 to about 1 percent of the total weight of thealcohol, including about 0.05 to about 1 percent, about 0.08 to about0.8 percent, about 0.1 to about 0.8 percent). The proton donor is anysuitable compound, such as hydrochloric acid, nitric acid, phosphoricacid, phosphonic acid, boric acid, sulfuric acid, adipic acid, benzene1,3,5 tricarboxylic acid, chlorosuccinic acid, cholinechloride,cis-aconitic acid, citramalic acid, citric acid, cyclobutane 1,1,3,3tetracarboxylic acid, cyclohexane 1,2,4,5 tetracarboxylic acid,cyclopentane 1,2,3,4 tetracarboxylic acid, diglycolic acid, fumaricacid, glutamic acid, glutaric acid, glyoxylic acid, isocitric acid,ketomalonic acid, lactic acid, maleic acid, malic acid, malonic acid,nitrilotriacetic acid, oxalacetic acid, oxalic acid, phytic acid,p-toluenesulfonic acid, salicylic acid, succinic acid, tartaric acid,tartronic acid, tetrahydrofuran 2,3,4,5 tetracarboxylic acid,tricarballylic acid, versene acids, 3-hydroxyglutaric acid,2-hydroxypropane, 1,3 dicarboxylic acid, glyceric acid, furan 2,5dicarboxylic acid, 3,4-dihydroxyfuran-2,5 dicarboxylic acid,3,4-dihydroxytetrahydrofuran-2,5-dicarboxylic acid, 2-oxo-glutaric acid,dl-glyceric acid, 2,5 furan-dicarboxylic acid, or mixtures thereof.Preferably the proton donor is citric acid, tartaric acid, malonic acid,and/or malic acid. More preferably, the proton donor is citric acid.

If desired, the composition can contain other components that include,for example, propylene glycol, a thickening agent (e.g., polyacrylicacid), a humectant (e.g., glycerine, aloe vera), an essential oil (e.g.,tea tree oil), fruit extract, fragrance (e.g., carbomer, aminomethylpropanol, isopropyl myristate, tocopheryl acetate), and/or dye (e.g.,blue 1, red 33, yellow 5). Depending on the carrier, polymers used, andpresence of additional components, the composition can be in anydesirable formulation, including a liquid, a cream, a gel, or a foam.

In a specific example, the composition comprises non-chemically modifiedlinear PEI, polyDADMAC, optionally citric acid, a carboxylated branchedPEI, and a water/alcohol carrier.

In an embodiment, the polyethylenimine-based polymer and second cationicpolymer form a crystalline miscible blend that results in a stabledispersion in the carrier. A miscible blend provides a clear crystallinesolution that is stable, i.e., from which there is no precipitation orfall-out. A miscible blend is different than a PEC. In addition, withoutwishing to be bound by any theory, it is believed that the crystallinenature of the polymers in the blend have a greater surface area and areable to provide more of an interface between the cationic polymer andmicrobial pathogens, both in solution and as a film.

The crystalline miscible blend can be prepared by any suitable method.In a specific example, a miscible blend of non-chemically modifiedlinear PEI and other cationic polymers can be prepared as follows. Anappropriate amount of water/PEI dispersion (e.g., about 4000 ppm PEI) isheated to a temperature that is slightly above the glass transitiontemperature of PEI (e.g., at least 1° C. above the glass transitiontemperature, at least about 2° C. above the glass transitiontemperature, at least about 3° C. above the glass transitiontemperature, at least about 4° C. above the glass transitiontemperature, at least about 5° C. above the glass transitiontemperature; including a temperature that ranges from 65-80° C. or from68-78° C. or from 70-75° C. or about 70° C., about 72° C., or about 74°C.). Next, an appropriate amount of a second cationic polymer (e.g.,polyDADMAC) solution with a lower pH (e.g., a pH of about 5-6, includinga pH of about 5, a pH of about 5.5, or a pH of about 6), is added. Thislower pH further helps to ensure that the PEI will remain in a solutionstate. After vigorous mixing, the solution is cooled to roomtemperature. Next, an appropriate amount (e.g., between 25 and 100 ppm)of an adhesion promoter (e.g., carboxylated branched PEI) is added.Carboxylated branched PEI is very basic, which will raise the pH of thesolution. The pH should be adjusted back to 6.5 so that the linear PEIdoes not solidify. Next, the solution is reheated to a temperature thatis slightly above the glass transition temperature of PEI (e.g., atleast 1° C. above the glass transition temperature, at least about 2° C.above the glass transition temperature, at least about 3° C. above theglass transition temperature, at least about 4° C. above the glasstransition temperature, at least about 5° C. above the glass transitiontemperature; including a temperature that ranges from 65-80° C. or from68-78° C. or from 70-75° C. or about 72°, or about 75° C.). Whilevigorously stirring the blend, an appropriate amount of alcohol isdrizzled in. The blend is continuously stirred while cooling to roomtemperature. The room temperature solution is then stirred for anadditional 24 hours.

The PEI-containing composition can have one or more bactericidal,virucidal, and/or germicidal properties and can, if desired, be used asan antimicrobial composition, in particular as a hand sanitizer, inaccordance with the tests, substrates, and/or methods described herein.Accordingly, provided is a method of disinfecting a surface comprisingapplying to the surface a composition comprising apolyethylenimine-based polymer, optionally a second cationic polymerselected from a polydiallyldialkylammonium salt, apoly(acrylamide-co-diallyldialkylammonium halide), chitosan, or acombination thereof, optionally a polyacid, optionally at least oneadhesion promoter, and a carrier. In an aspect of this method, acomposition comprising non-chemically modified, linear PEI, optionally apolyacid, and a carrier comprising water and alcohol is used as a handsanitizer that is particularly effective against non-enveloped viruses.

Because linear PEI is pH- and temperature-sensitive, special techniqueswere developed to create a stable, crystal clear, non-sticky, handsanitizer blend comprising a linear PEI colloidal dispersion. In aspecific example, a method of preparing a hand sanitizer compositionincludes the following steps: At room temperature, an appropriate amountof linear PEI in water was stirred vigorously to create a PEIdispersion. Under vigorous stirring, the PEI in the dispersion was thenprotonated with an appropriate acid, thereby bringing the pH to 6 andresulting in a clear liquid. The clear liquid was then brought to atemperature that is slightly above the glass transition temperature ofPEI (e.g., at least 1° C. above the glass transition temperature, atleast about 2° C. above the glass transition temperature, at least about3° C. above the glass transition temperature, at least about 4° C. abovethe glass transition temperature, at least about 5° C. above the glasstransition temperature; including a temperature that ranges from 65-80°C. or from 68-78° C. or from 70-75° C. or about 70° C., about 72° C., orabout 74° C.). An appropriate amount of alcohol was then drizzled in soas to maintain the temperature of the clear liquid at about 65° C. Theclear hand sanitizer mixture was taken off the heat to avoid excessivealcohol evaporation and then stirred for several hours (e.g., at least 2hours, at least 3 hours, at least 4 hours, or at least 5 hours) whilecovered.

An antimicrobial composition of the present invention meets or exceedsat least one of the following antimicrobial tests:

(i) a germicidal spray test according to ASTM E1153 that meets the EPArequirement of log 3 reduction for viruses and a log 5 reduction forbacteria,

(ii) a suspension test according to ASTM E1052-96 (2002) or ASTM E2315(2016),

(iii) a film formed from the composition kills

-   -   (iii-a) at least 95% of log 5 population of a gram positive or        gram negative bacteria in 30 minutes,    -   (iii-b) at least 95% of log 4 population of an enveloped virus        within 30 minutes of contact of contact,    -   (iii-c) at least 95% of a non-enveloped virus within 30 minutes        of contact, and/or    -   (iii-d) at least 94% of a log 4 population of Clostridium        difficile bacteria within 24 hours of contact,        in accordance with JIS Z 2801 (2006) test for antimicrobial        activity, or a modified version of such test as described        herein,

(iv) a film formed from the composition has a value of 2 or lessaccording to International Organization for Standardization (ISO)10993-5 in vitro cytotoxicity test,

(v) a durability test selected from either (v-a) a film formed from thecomposition kills at least 99.9% of gram-positive bacteria andgram-negative bacteria according to EPA Protocol #01-1A residualself-sanitizing activity test, or (v-b) waiting 7 days after filmformation, a film formed from the composition kills at least 95% ofgram-positive bacteria and gram-negative bacteria, or enveloped andnon-enveloped viruses according a modified version of Protocol #01-1Aresidual self-sanitizing activity test, as described herein.

Test (i) refers to ASTM E1153, the entire contents of which areincorporated by reference, is a germicidal spray test (i.e., “kill now”claim) that is used to evaluate the antimicrobial efficacy of one-stepcleaner-sanitizer formulations recommended for use on lightly soiled,inanimate, nonporous, non-food contact surfaces. In ASTM E1153 (lastrevision in 2014), the antimicrobial efficacy of sanitizers is tested onprecleaned, inanimate, hard, nonporous, non-food contact surfacesagainst Staphylococcus aureus, Klebsiella pneumoniae, Enterobacteraerogenes, or a combination thereof.

Germicidal spray test results related to an inventive miscible blendformulation are set forth in Table 3 along with a comparison with threecommercially available products. MS2, MRSA, and E. coli germicidal spraytests were conducted with two cationic polymer compositions. CompositionA was a miscible blend comprising 3000 ppm non-chemically modified,linear PEI, 3000 ppm polyDADMAC, 25 ppm carboxylated branched PEI, 35%ethanol, and the balance of water. Composition B was a miscible blendcomprising 200 ppm of non-chemically modified, linear PEI, 200 ppmpolyDADMAC, 25 ppm branched PEI carboxylated, 70% ethanol, and thebalance in water (operational pH of about 7.6).

TABLE 3 Inventive antimicrobial compositions Disinfectant only A BProduct name OPTI-CIDE 3 ™ CAVICIDE1 ™ ASEPTICARE ™ Time to Kill(Biotrol) (Metrex) TB + II (Ecolab) Active ingredients Alcohol-QuatAlcohol-Quat Alcohol-Quat Alcohol-Polymer charge Non-enveloped Virus MS2(proxy for non- No No 6 min 2 min 5 min enveloped virus)- testingchallenge 99.99% Gram Positive Bacteria MRSA-testing 2 min 1 min 6 min 2min 30 sec challenge 99.999% (with 5% soil load) Gram Negative BacteriaE. Coli-testing 2 min 1 min 6 min 2 min 30 sec challenge 99.999% (with5% soil load)

The results in Table 3 demonstrate that Composition B contained 400 ppmof cationic polymer and passed the MS2 germicidal spray test within 5minutes of contact, which is notable since the EPA's maximum ppmstandard for quaternary ammonium compounds in accordance with ASTM E1153is 400 ppm. Moreover, the antimicrobial efficacy of some quaternaryammonium compounds is significantly diminished by soil or an organicload. As seen in Table 3, Composition B was also highly effectiveagainst MRSA and E. Coli spray tests (ASTM E1153) in the presence of a5% soil load.

Test (ii) is a suspension test in accordance with ASTM E1052-96 (2002)or ASTM E2315 (2016) to determine the effectiveness of an antimicrobialsolution that is in the form of a suspension against specific viruses,such as adenovirus, coronavirus, influenza viruses, rhinovirus, androtavirus. An aliquot of the test substance is inoculated with the testvirus and held for the requested exposure time. At each pre-determinedexposure time, an aliquot is removed, neutralized by serial dilution,and assayed for viral infectivity by an assay method specific for thetest virus. Appropriate virus, test substance cytotoxicity, andneutralization controls are run concurrently. The percent and logreduction in viral infectivity are calculated as compared to thecorresponding virus control. ASTM E1052-96 (2002) and ASTM E2315 (2016)are most appropriate for an antimicrobial composition that is asuspension, such as a hand sanitizer composition.

For test (iii), the ability for a film formed from the antimicrobialcomposition to kill gram positive and gram negative bacteria and anenveloped virus, a non-enveloped virus, and/or Clostridium difficilebacteria can be tested in accordance with the conditions set forth inJIS Z 2801 (2006 version, updated in 2010), which is known as theJapanese Industrial Standard Test for Antimicrobial Activity andEfficacy in Antimicrobial Products, the entire contents of which areincorporated by reference. In particular, in accordance with JIS Z 2801(2006) or a modified version thereof, as described herein, a film formedfrom an antimicrobial composition of the present invention kills:(iii-a) at least 95% of log 5 population of a gram positive or gramnegative bacteria in 30 minutes, (iii-b) at least 95% of log 4population of an enveloped virus within 30 minutes of contact, (iii-c)at least 95% of a non-enveloped virus within 30 minutes of contact,and/or (iii-d) at least 94% of a log 4 population of Clostridiumdifficile bacteria within 24 hours of contact. In a preferredembodiment, a film formed from an antimicrobial composition of thepresent invention meets 2 or more, 3 or more, or all 4 of each of therequirements (iii-a)-(iii-d).

For example, under the conditions of this test, the JIS Z 2801 protocoldemonstrated a log 4 reduction of E. coli on a film comprising onlypolyDADMAC after 30 minutes, against a log 5 challenge (Table 4).

TABLE 4 Escherichia coli % Anti- ATCC 8739 Reduction microbial SampleIdentification cfu/sample after 30 min activity Untreated control 2.20 ×10⁵ n/a n/a 1 (pDADMAC, 12K ppm, <15 >99.99% >4.17 160 ppm titanate(LICA ™ 09), 50% EtOH, pH 3.4) * cfu/sample—colony forming units persample recovered

The same film demonstrated a log 4 reduction of MRSA after 10 minutes,but with a log 7 challenge (Table 5).

TABLE 5 Methicillin Resistant % Anti- Staphylococcus aureus Reductionmicrobial Sample Identification ATCC 33591 cfu/sample after 10 minactivity Untreated control 7.55 × 10⁶ n/a n/a 1 (pDADMAC, 12K 7.20 × 10²99.99% 4.02 ppm, 160 ppm titanate (LICA ™ 09), 50% EtOH, pH 3.4) *cfu/sample—colony forming units per sample recovered

An additional self-sanitizing (“kill later”) test was devised for aresidual self-sanitizing film that is believed to be more practical fora realistic utility, particularly when testing antiviral activity. Thistest is based on the assumption that in a real world application, anantimicrobial residual self-sanitizing film would not be covered. Thistest modifies JIS Z 2801 (2006 version, which was updated in 2010) bynot requiring the inoculated film to be covered and starting the testtime after the inoculum dries. Test results using modified JIS Z 2801are set forth in Table 6 for determining the lysis of MS2 on a filmcreated from a non-toxic, miscible blend of 3000 ppm non-chemicallymodified, linear PEI, 3000 ppm polyDADMAC, 79% ethanol, 25 ppmcarboxylated branched PEI, and the balance water. The “kill later” datafor gram positive and gram negative bacteria were generated using thestandard JIS test.

TABLE 6 % killed % killed % killed % killed % killed Pathogen 1 min 5min 10 min 15 min 20 min Non-enveloped Virus MS2-testing 99.99% 95.0099.38 99.96 challenge Gram Positive Bacteria MRSA-testing 99.83 99.99challenge 99.999% Gram Negative Bacteria E. Coli-testing 99.54 99.99challenge 99.999%

Moreover, JIS Z 2801 (2006 version, which was updated in 2010) can bemodified when testing against Clostridium difficile bacteria by testingunder lighted conditions and increasing the sample surface area from1600 mm² to 2500 mm².

Test (iv) is directed to ISO 10993-5 (last updated in 2009), the entirecontents of which are incorporated by reference, in which the in vitrocytotoxicity of medical device materials is tested. The method isdirected to the incubation of cultured cells in contact with a deviceand/or extracts of a device either directly or through diffusion. Inparticular, the test article, positive and negative controls areextracted according to the method ISO 10993-12. The original extract isserially diluted and 5 concentrations are used for testing. L-929 cells(mouse, C3H/An, connective tissue) are treated with extracts of thesample, reagent control, and either negative control or positivecontrol. Triplicate plates are prepared for each treatment. The cellsare incubated for 24 hours and observed microscopically for cytotoxiceffects. Cultures are observed under microscopy and graded forreactivity using a 0 to 4 scale (“4” means severely cytotoxic; “3” meansmoderately cytotoxic; “2” means mildly cytotoxic; “1” means slightlycytotoxic, and “0” means non-cytotoxic). Test article meets therequirement of the test when results are less than or equal to a gradeof 2 (i.e., 0, 1, or 2).

A residual self-sanitizing film formed from a PEC, a miscible blend, orindividual cationic polymers, as described herein, is non-leaching, andthus passes the ISO 10993-5 (2009 version) in vitro cytotoxicity testwith a score of 0, as shown by the following test results in Table 7.

TABLE 7 Sample Identification Cytotoxic Grade Reactivity 1 (pDADMAC PECwith PAAS, 3/6 0 Non-cytotoxic highly diluted, 6K ppm, no adhesionpromoter, pH 7.2) 2 (pDADMAC PEC with PAAS, 3/6 0 Non-cytotoxic highlydiluted, 6K ppm, 3 drops titanate (LICA ™ 09), pH 7.2) 3 (pDADMAC PECwith PAAS, 3/6 0 Non-cytotoxic highly diluted, 4K ppm, 3 titanate(LICA ™ 09), pH 7.2) 4 (50% pDADMAC, 50% PEI, 0 Non-cytotoxic titanate,4K ppm, 20% EtOH, pH 7) 5 (linear, non-modified PEI, 4K ppm, 0Non-cytotoxic pH 6) Negative control 0 Non-cytotoxic Reagent control 0Non-cytotoxic Positive controls 3/4 Moderately/ severely cytotoxic

Test (v) is directed to Protocol #01-1A, commonly known as “the Cloroxtest,” which is a method approved by the EPA for measuring long-termsanitization claims (i.e., “kill later” durability claim). Protocol#01-1A, the entire contents of which are incorporated by reference,measures the residual self-sanitizing activity of dried chemicalresidues (films) on inanimate, hard, non-porous surfaces against onlybacteria: Staphylococcus aureus, Klebsiella pneumoniae, and/orEnterobacter aerogenes. In particular, surfaces are inoculated, treatedwith test product, allowed to dry, then abraded under alternating wetand dry conditions, which are interspersed with several re-inoculations.At the end of the study and at least 24 hours later, the ability of thetest surfaces to kill 99.9% of microorganisms within 5 minutes ismeasured. To pass this test, a film formed from the composition mustmaintain its antimicrobial efficacy between, and after, 12 alternatingwet and dry rubs with a weighted cloth.

A modified version of the EPA's durability test, Protocol #01-1A can beused. It is believed that a modified protocol is more appropriate forevaluating residual self-sanitizing films formed from an inventiveantimicrobial composition because Protocol #01-1A was designed forproducts that depend on killing microbes by releasing germicidalchemicals from a film and which deplete over time. The inventiveantimicrobial composition does not require germicidal chemicals, butrather comprises charged, cationic polymers whose killing mechanism isnot believed to deplete over time. The modified test consists of dailysubmitting the film to three rubs (one dry, one wet, one dry) using theEPA #01-1A protocol weight and cycle time. This modified test capturesthe antimicrobial effectiveness of an antimicrobial composition overdays, compared to the single 24 hour measurement of the standardProtocol #01-1A. Passing the modified test will require that after 4 to7 days, the polymer-based film will continue to demonstrate at least a95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, at least 99.9%) reduction of gram-positive, gram-negativebacteria, enveloped virus, and/or non-enveloped virus on glass orstainless steel substrates.

The invention also provides a method of killing microbes on a surfacecomprising applying to the surface an antimicrobial compositioncomprising a cationic polymer (by itself or encapsulated in a PEC), atleast one adhesion promoter (e.g., a titanate, a carboxylated branchedPEI), optionally organic and/or inorganic particles that arephotocatalyically active in visible light, and a carrier, each of thesecomponents are as described herein. The method can include disinfectinga surface, providing a residual self-sanitizing film, or both. The term“microbe” includes any single cell or multicellular organism, such asbacteria, viruses, fungi, archaea, and protists (e.g., algae, amoebas,protozoa). As used herein, the term “applying” refers to any suitabletechnique used to transfer the antimicrobial composition to a surface.For example, techniques for applying can be, but are not limited to,brushing, rolling, spraying, wiping, mopping, pouring, painting,absorbing, adsorbing, imbibing, soaking, saturating, permeating,immersing, and a combination of these methods.

Further provided is a method of killing microbes on a surface (e.g.,disinfecting a surface, providing a residual self-sanitizing film, orboth) comprising applying to the surface an antimicrobial compositioncomprising a high molecular weight (preferably an ultra-high molecularweight) polydiallyldimethylammonium salt (e.g.,polydiallyldimethylammonium halide) and a carrier, as described herein.The antimicrobial composition of this embodiment can further comprise(i) a polyethylenimine-based polymer, chitosan, or a combinationthereof, and/or (ii) an anionic polymer, and/or (iii) organic and/orinorganic particles that are photocatalyically active in visible light,and/or (iv) at least one adhesion promoter (e.g., a titanate, acarboxylated branched PEI), and/or (v) at least one salt. Each of theseoptional components is as described herein.

Once applied to the surface, the carrier, as described herein, in thecomposition evaporates to leave an antimicrobial residualself-sanitizing film on the surface. The antimicrobial residualself-sanitizing film renders the surface bactericidal, virucidal, and/orgermicidal. As used herein, the term “renders the surface bactericidal,virucidal, and/or germicidal” refers to reducing (e.g., eliminating,killing, or preventing and/or inhibiting growth) the presence ofbacteria, viruses, and/or germs (including a fungus, such as Aspergillasbrasliensis) to any suitable degree. As used herein, the term “anysuitable degree” refers to 50% reduction or more, including 60%reduction or more, 70% reduction or more, 80% reduction or more, 90%reduction or more, 92% reduction or more, 94% reduction or more, 95%reduction or more, 97% reduction or more, 98% reduction or more, 99%reduction or more, or 99.5% elimination or more.

In accordance with this embodiment, the invention provides a coatedsurface comprising a surface (e.g., a surface of a substrate) and anantimicrobial residual self-sanitizing film, as described herein, thatis applied to the surface. The resulting film provides a non-leachingsurface that is not easily removed. In most embodiments, theantimicrobial residual self-sanitizing film is not covalently bound tothe surface (e.g., surface of the substrate).

The surface that is rendered bactericidal, virucidal, and/or germicidalcan be of any suitable material, including a biocompatible material. Thesurface can be used in or derived from any suitable form, such as, forexample, a powder, dust, an aggregate, an amorphous solid, a sheet, afiber, a tube, a fabric, or the like. In embodiments, the surfacecomprises metal, glass, fiberglass, silica, sand, wood, fiber, naturalpolymer, synthetic polymer, plastic, rubber, ceramic, porcelain, stone,marble, cement, a human or animal body (e.g., skin), or any hybrid,alloy, copolymer, blend, or combination thereof.

Metal surfaces suitable for use in the invention include, for example,stainless steel, nickel, titanium, tantalum, aluminum, copper, gold,silver, platinum, zinc, nickel titanium alloy (nitinol), an alloy ofnickel, chromium, and iron (INCONEL™, Special Metals, Corporation,Elkhart, Ind.), iridium, tungsten, silicon, magnesium, tin, galvanizedsteel, hot dipped galvanized steel, electrogalvanized steel, annealedhot dipped galvanized steel, alloys of any of the foregoing metals,coatings containing any of the foregoing metals, and combinationsthereof.

Glass surfaces suitable for use in the invention include, for example,soda lime glass, strontium glass, borosilicate glass, barium glass,glass-ceramics containing lanthanum, fiber glass, and combinationsthereof.

Silica surfaces suitable for use in the invention include, for example,quartz, fused quartz, crystalline silica, fumed silica, silica gel,silica aerogel, and mixtures thereof.

Sand surfaces suitable for use in the invention include, for example,sand comprised of silica (e.g., quartz), calcium carbonate (e.g.,aragonite), and mixtures thereof. The sand can comprise othercomponents, such as minerals (e.g., magnetite, chlorite, glauconite,gypsum, olivine, garnet), metal (e.g., iron), shells, coral, limestone,and/or rock.

Suitable wood surfaces include, for example, hard wood and soft wood,and materials engineered from wood, wood chips, or fiber (e.g., plywood,oriented strand board, laminated veneer lumber, composites, strandlumber, chipboard, hardboard, medium density fiberboard), andcombinations thereof. Types of wood include alder, birch, elm, maple,willow, walnut, cherry, oak, hickory, poplar, pine, fir, andcombinations thereof.

Fiber surfaces suitable for use in the invention include, for example,natural fibers (e.g., derived from an animal, vegetable, or mineral) andsynthetic fibers (e.g., derived from cellulose, mineral, or polymer).Suitable natural fibers include cotton, hemp, jute, flax, ramie, sisal,bagasse, wood fiber, silkworm silk, spider silk, sinew, catgut, wool,sea silk, wool, mohair, angora, and asbestos. Suitable synthetic fibersinclude rayon (e.g., lyocell), modal, and metal fiber (e.g., copper,gold, silver, nickel, aluminum, iron), carbon fiber, silicon carbidefiber, bamboo fiber, seacell, nylon, polyester, polyvinyl chloride fiber(e.g., vinyon), polyolefin fiber (e.g., polyethylene, polypropylene),acrylic polyester fiber, aramid (e.g., TWARON™, KEVLAR™, or NOMEX™),spandex, and combinations thereof.

Natural polymer surfaces suitable for use in the invention include, forexample, a polysaccharide (e.g., cotton, cellulose), shellac, amber,wool, silk, natural rubber, a biopolymer (e.g., a protein, anextracellular matrix component, collagen), and combinations thereof.

Synthetic polymer surfaces suitable for use in the invention include,for example, polyvinylpyrrolidone, acrylics,acrylonitrile-butadiene-styrene, polyacrylonitrile, acetals,polyphenylene oxides, polyimides, polystyrene, polypropylene,polyethylene, polytetrafluoroethylene, polyvinylidene fluoride,polyvinyl chloride, polyethylenimine, polyesters, polyethers, polyamide,polyorthoester, polyanhydride, polysulfone, polyether sulfone,polycaprolactone, polyhydroxy-butyrate valerate, polylactones,polyurethanes, polycarbonates, polyethylene terephthalate, as well ascopolymers and combinations thereof.

Typical rubber surfaces suitable for use in the invention include, forexample, silicones, fluorosilicones, nitrile rubbers, silicone rubbers,polyisoprenes, sulfur-cured rubbers, butadiene-acrylonitrile rubbers,isoprene-acrylonitrile rubbers, and combinations thereof.

Ceramic surfaces suitable for use in the invention include, for example,boron nitrides, silicon nitrides, aluminas, silicas, combinationsthereof, and combinations thereof.

Stone surfaces suitable for use in the invention include, for example,bauxite, calcite, feldspar, gypsum, slate, granite, quartz, quartzite,limestone, dolostone, sandstone, marble, soapstone, serpentine andcombinations thereof.

For purposes of the present invention, animal bodies include, but arenot limited to, the order Rodentia (e.g., mice), the order Logomorpha(e.g., rabbits), the order Camivora (e.g., Felines (cats) and Canines(dogs)), the order Artiodactyla (e.g., Bovines (cows) and Swines(pigs)), the order Perssodactyla (e.g., Equines (horses)), the orderPrimates, Ceboids, or Simioids (e.g., monkeys), the class Ayes (e.g.,birds), the class of Phylum Arthropoda (e.g., insects), the class ofPisces (e.g., fish), or the order Anthropoids (e.g., humans and apes).Typically skin (including intact skin, wounded or broken skin, and/orskin that is otherwise damaged, by for example, a burn) and/or mucosaltissue (e.g., oral, nasal, ocular, or genital tissue) of the animal bodyserves as the surface suitable for application of the antimicrobialcomposition. The skin and/or mucosal tissue can be associated with anypart of the animal body, including the limbs, tail, abdomen, chest,head, neck, face, genital area (e.g., udder), buttocks, or back. Ingeneral, the type and amount of components of the antimicrobialcomposition will be selected to ensure biocompatibility, to minimizetoxicity, to minimize irritation, and/or have a desired level of surfacetack and/or adhesiveness of the formed film.

The surface typically is a component of a larger structure. For example,the surface can be part of a substrate, such as a medical device,diagnostic equipment, implant, glove, mask, curtain, mattress, sheets,blankets, gauze, dressing, tissue, surgical drape, tubing, surgicalinstrument, safety gear, fabric, apparel item, floor, handles, wall,sink, shower or tub, toilet, furniture, wall switch, toy, athleticequipment, playground equipment, shopping cart, countertop, appliance,railing, door, air filter, pipe, utensil, dish, cup, container, objectdisplay container, food, food display container, food package, foodprocessing equipment, food handling equipment, food transportationequipment, food vending equipment, food storage equipment, foodpackaging equipment, plant, phone, cell phone, remote control, computer,mouse, keyboard, touch screen, leather, cosmetic, cosmetic makingequipment, cosmetics storage equipment, cosmetics packaging equipment,personal care item, personal care item making equipment, personal carestorage equipment, personal care packaging equipment, animal care item,animal care item making equipment, veterinary equipment, powder, cream,gel, salve, eye care item, eye care item making equipment, contact lens,glasses, eye care storage equipment, contact lens case, jewelry, jewelrymaking equipment, jewelry storage equipment, animal housing, farmingequipment, animal food handling equipment, animal food storage space,animal food storage equipment, animal food container, air vehicle, landvehicle, air processing equipment, air filter, water vehicle, waterstorage space, water storage equipment, water processing equipment,water storage container, water filter, hand, hair, foot, leg, arm,torso, head, or animal body part, pharmaceuticals display container,pharmaceuticals package, pharmaceuticals processing equipment,pharmaceuticals handling equipment, pharmaceuticals transportationequipment, pharmaceuticals vending equipment, pharmaceuticals,pharmaceuticals storage equipment, pharmaceuticals packaging equipment.

A “medical device” includes any device having surfaces that contacttissue, blood, or other bodily fluids in the course of their use oroperation, which are found on or are subsequently used within a mammal(e.g., a human) Medical devices include, for example, extracorporealdevices for use in surgery, such as blood oxygenators, blood pumps,blood storage bags, blood collection tubes, blood filters includingfiltration media, dialysis membranes, tubing used to carry blood and thelike which contact blood which is then returned to the patient ormammal. Medical devices also include endoprostheses implanted in amammal (e.g., a human), such as vascular grafts, stents, pacemakerleads, surgical prosthetic conduits, heart valves, and the like, thatare implanted in blood vessels or the heart. Medical devices alsoinclude devices for temporary intravascular use such as catheters, guidewires, amniocentesis and biopsy needles, cannulae, drainage tubes,shunts, sensors, transducers, probes and the like which are placed intothe blood vessels, the heart, organs or tissues for purposes ofmonitoring or repair or treatment. Medical devices also includeprostheses such as artificial joints such as hips or knees as well asartificial hearts. In addition, medical devices include penile implants,condoms, tampons, sanitary napkins, ocular lenses, sling materials,sutures, hemostats used in surgery, antimicrobial materials, surgicalmesh, transdermal patches, and wound dressings/bandages.

The “diagnostic equipment” includes any device or tool used to diagnoseor monitor a medical condition. Examples include an ultrasound, magneticresonance imaging (MRI) machine, positron emission tomography (PET)scanner, computed tomography (CT) scanner, ventilator, heart-lungmachine, extracorporeal membrane oxygenation (ECMO) machine, dialysismachine, blood pressure monitor, otoscope, ophthalmoscope, stethoscope,sphygmomanometer, blood pressure cuff, electrocardiograph, thermometer,defibrillator, speculum, sigmoidoscope, and anoscope.

The “surgical instrument” includes any tool or device used forperforming surgery or an operation. Examples include a scalpel, lancet,trocar, hemostat, grasper, forceps, clamp, retactor, distractor,positioner, tracheotome, dilator, stapler, irrigation needle, injectionneedle, drill, scope, endoscope, probe, ruler, and caliper.

“Safety gear” includes devices used to protect a person, animal, orobject. Examples of “safety gear” are a mask, face shield, visor,goggles, glasses, gloves, shoe covers, foot guard, leg guard, belt,smock, apron, coat, vest, raingear, hat, helmet, chin strap, hairnet,shower cap, hearing protection (ear plugs, ear muffins, hearing bands),respirator, gas mask, supplied air hood, collar, leash, and first aidkit.

“Fabric” includes any type of suitable fabric, such as bedding,curtains, towels, table coverings, protective sheeting, and dish cloths.

An “apparel item” includes an item of clothing, footwear, or other itemsomeone would wear on his/her person. Examples include a uniform, coat,shirt, pants, waders, scrubs, socks, shoe or boot liner, an insole,gloves, hats, shoes, boots, and sandals.

The surface can be part of a building structure or an item that can befound in a building structure, such as a floor, wall, an appliance(e.g., a refrigerator, oven, stove, dishwasher, washing machine, clothesdryer, furnace, water heater, air conditioner, heater), sink, shower ortub, toilet, furniture (e.g., mattress, couch, sofa, chair, table,shelf, mantle, bed, dresser), countertop, railing, air filter, airprocessing equipment, water processing equipment, water filter, pipe,door, handle, light, light switch, thermostat, sprinkler, airconditioner evaporator and/or condenser.

The surface can also be a toy or athletic equipment, including exerciseequipment, playground equipment, or a pool.

The surface can be a utensil (e.g., knife, fork, spoon, ladle, spatula,whisk, etc.), a dish (e.g., a food storage container, a food servingpiece, etc.), a food package (e.g., a bag, a box, foil, plastic wrap),or other item that comes in contact with food (e.g., a cutting board,food display container, food processing equipment, food handlingequipment, food transportation equipment, food vending equipment, animalfood handling equipment, animal food storage space, food storageequipment, animal food container, animal food storage equipment). Thesurface can be part of food processing equipment, such as foodprocessing tanks, stirrers, conveyor belts, knives, grinders, packagingmachines, labeling machines, etc.

The “food” is any food in which it would be desirable to provide with anantimicrobial residual self-sanitizing film. In such embodiments, theantimicrobial residual self-sanitizing film and the composition thereofshould be nontoxic for human and animal consumption. The “food” can be,e.g., any fruit, vegetable, meat, or egg.

The “plant” is any suitable plant, including an angiosperm (a floweringplant), gymnosperm (a seed-producing plant), a conifer, fern, and moss.Suitable angiosperms are from the amborella (e.g., Amborella trichopodaBaill), nymphaeales (e.g., water lily), austrobaileyales (e.g., Illiciumverum), chloranthales (e.g., from the genus ascarina, chloranthus,hedyosmum, or sarcandra), magnoliids (e.g., magnolia, bay laurel, blackpepper), monocots (e.g., grasses, orchids, palms), ceratophyllum (e.g.,aquatic plants), or eudicots (e.g., sunflower, petunia, apple) groups.Suitable gymnosperms are from the subclass cycadidae, ginkgoidae,gnetidae, or pinidae.

The surface can be part of an electronic device, such as a phone, cellphone, remote control, computer, mouse, keyboard, and touch screen.

The surface can further be part of a cosmetic (e.g., eye shadow,eyeliner, primer, foundation, lipstick, lip gloss, blush), cosmeticmaking equipment, cosmetic storage equipment, cosmetic packagingequipment, a personal care item (e.g., cream, gel, salve, lip balm, bodysoap, facial soap, lotion, cologne, perfume, antiperspirant, deodorant,facial tissue, cotton swabs, cotton pads, mouthwash, toothpaste, nailpolish, shampoo, conditioner, hairspray, talcum powder, shaving cream,contact lens, contact lens case, glasses), personal care item makingequipment, personal care storage equipment, personal care packagingequipment, jewelry (e.g., necklace, ring, earring, bracelet, watch),jewelry making equipment, or jewelry storage equipment.

The “animal care item” and “veterinary equipment” can be any productused in a setting that includes animals, such as a house, boardinghouse, or veterinary hospital. Of course, veterinary equipment can beused at a location outside of a hospital setting. Animals are anyanimals that are typically considered pets, non-pets, boarded, treatedby a veterinarian, and animals in the wild. Examples include a dog, cat,reptile, bird, rabbit, ferret, guinea pig, hamster, rat, mouse, fish,turtle, horse, goat, cattle, and pigs. Suitable animal care itemsinclude the personal care items described herein, toys, bed, crate,kennel, carrier, bowl, dish, leash, collar, litterbox, and groomingitems (e.g., clippers, scissors, a brush, comb, dematting tool, anddeshedding tool). Suitable veterinary equipment includes any of themedical devices and surgical instruments described herein and otherequipment, such as a table, tub, stretcher, sink, scale, cage, carrier,and leash.

The “animal housing” can be any suitable housing, such as a coop,stable, shelter, grab bag shelter, hutch, barn, shed, pen, nestbox,feeder, stanchion, cage, carrier, or bed.

The “farming equipment” is any device used in an agricultural setting,including a farm or ranch, particularly a farm or ranch that housesanimals, processes animals, or both. Animal livestock that can be housedor processed as described herein and include, e.g., horses, cattle,bison, and small animals such as poultry (e.g., chickens, quails,turkeys, geese, ducks, pigeons, doves, pheasants, swan, ostrich,guineafowl, Indian peafowl, emu), pigs, sheep, goats, alpacas, llamas,deer, donkeys, rabbits, and fish. Examples of farming equipment includeas a wagon, trailer, cart, barn, shed, fencing, sprinkler, shovel,scraper, halter, rope, restraining equipment, feeder, waterer, trough,water filter, water processing equipment, stock tank, fountain, bucket,pail, hay rack, scale, poultry flooring, egg handling equipment, a barncurtain, tractor, seeder, planter, plow, rotator, tiller, spreader,sprayer, agitator, sorter, baler, harvester, cotton picker, thresher,mower, backhoe loader, squeeze chute, hydraulic chute, head chute, headgate, crowding tub, corral tub, alley, calving pen, calf table, andmilking machine.

The surface can be part of a vehicle, such as an air vehicle, landvehicle, or water vehicle. Suitable vehicles include a car, van, truck,bus, ambulance, recreational vehicle, camper, motorcycle, scooter,bicycle, wheelchair, train, streetcar, ship, boat, canoe, submarine, anunmanned underwater vehicle (UUV), a personal water craft, airplane,jet, helicopter, unmanned autonomous vehicle (UAV), and hot air balloon.

If desired, the surface to which the antimicrobial residualself-sanitizing film has been applied can be regenerated by removing theantimicrobial residual self-sanitizing film, since the film typically isnot covalently bonded to the surface. The removing step can be performedby any suitable method, such as washing or rinsing with a solvent (e.g.,water and/or alcohol). Thus, the antimicrobial coating on a surface(e.g., the surface of a substrate) described herein can be consideredtemporary (e.g., removable). In an embodiment, the antimicrobialresidual self-sanitizing film is water soluble and is removable withwater (e.g., hot soapy water).

The antimicrobial residual self-sanitizing film renders the surfacebactericidal against any suitable bacteria to any suitable degree. Inother words, an antimicrobial composition of the present invention canform an antimicrobial residual self-sanitizing film on a surface (e.g.,the surface of a substrate) that kills at least 75% (e.g., at least 80%,at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%)of bacteria that comes in contact with the antimicrobial residualself-sanitizing film. For example, the bacteria can be, for example,Staphylococcus aureus, gram positive methicillin-resistantStaphylococcus aureus (MRSA), Staphylococcus saprophyticus, Pseudomonasaeruginosa, Listeria monocytogenes, Klebsiella pneumoniae, Streptococcuspneumoniae, Streptococcus pyogenes, Streptococcus agalactiae,Haemophilus influenzae, Helicobacter pylori, Salmonella, Shigella,Clostridium, Enterobacter aerogenes, gram negative Escherichia coli,Clostridium difficile, or a combination thereof. In certain embodiments,the antimicrobial composition is effective in reducing (e.g.,eliminating, killing, or preventing and/or inhibiting growth) grampositive methicillin-resistant Staphylococcus aureus (MRSA), gramnegative Escherichia coli (ATCC 8739), Clostridium difficile (ATCC43598), or a combination thereof.

In an aspect of the invention, an antimicrobial residual self-sanitizingfilm formed from an antimicrobial composition described herein rendersthe surface bactericidal against gram positive methicillin-resistantStaphylococcus aureus (MRSA) bacteria. Preferably, the antimicrobialresidual self-sanitizing film kills at least 95% (e.g., at least 96%, atleast 97%, at least 98%, at least 99%) of a log 5 population of grampositive methicillin-resistant Staphylococcus aureus (MRSA) bacteriawithin 30 minutes (e.g., within 20 minutes, within 15 minutes, within 10minutes, within 5 minutes) of contact. In a particularly preferredembodiment, the antimicrobial residual self-sanitizing film kills atleast 99.8% of a log 5 population of gram positive methicillin-resistantStaphylococcus aureus (MRSA) bacteria within 5 minutes of contact.

In another aspect of the invention, an antimicrobial residualself-sanitizing film formed from an antimicrobial composition describedherein renders the surface bactericidal against gram negativeEscherichia coli (ATCC 8739) bacteria. In particular, the antimicrobialresidual self-sanitizing film kills at least 95% (e.g., at least 96%, atleast 97%, at least 98%, at least 99%) of a log 5 population of gramnegative Escherichia coli (ATCC 8739) bacteria within 30 minutes (e.g.,within 20 minutes, within 15 minutes, within 10 minutes, within 5minutes) of contact. In a preferred embodiment, the antimicrobialresidual self-sanitizing film kills at least 99.7% of a log 5 populationof gram negative Escherichia coli (ATCC 8739) bacteria within 5 minutesof contact.

In yet another aspect of the invention, an antimicrobial residualself-sanitizing film formed from an antimicrobial composition describedherein renders the surface bactericidal against Clostridium difficile(ATCC 43598) bacteria. More specifically, the antimicrobial residualself-sanitizing film kills at least 75% (e.g., at least 80%, at least85%, at least 90%, at least 95%, at least 97%, at least 98%, at least99%) of a log 4 population of Clostridium difficile (ATCC 43598)bacteria within 24 hours of contact (e.g., within 18 hours, within 12hours, within 10 hours, within 8 hours, within 6 hours) of contact. In apreferred embodiment, the antimicrobial residual self-sanitizing filmkills at least 99.7% of a log 4 population of Clostridium difficile(ATCC 43598) bacteria within 8 hours of contact.

Viruses are much more difficult to kill, especially non-envelopedviruses, e.g., norovirus, rotavirus, adenovirus, and poliovirus.Generally, the only way to kill an array of non-enveloped viruses iswith an abundance of very harsh chemicals such as hypochlorite, acidsand peroxides, all of which are extremely cytotoxic. Remarkably, thetechnology described in the present invention is capable of formingantimicrobial residual self-sanitizing films that kill non-envelopedviruses. Accordingly the present invention provides an antimicrobialresidual self-sanitizing film formed from an antimicrobial compositiondescribed herein that renders a surface virucidal against any suitablevirus to any suitable degree, such as, reducing (e.g., eliminating,killing, or preventing and/or inhibiting growth) at least 75% (e.g., atleast 80%, at least 85%, at least 90%, at least 92%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or atleast 99.5%) of the virus. In a particular example, an antimicrobialresidual self-sanitizing film formed from an antimicrobial compositiondescribed herein renders the surface virucidal against at least oneenveloped virus (e.g., chickenpox virus, influenza, herpes simplex,severe acute respiratory syndrome (SARS), flavivirus, togavirus) ornon-enveloped virus (e.g., levivirus, norovirus, rotavirus, adenovirus,parvovirus, and poliovirus).

In another aspect of the invention, an antimicrobial residualself-sanitizing film formed from an antimicrobial composition describedherein renders the surface virucidal against influenza A (e.g., H1N1,H1N2, and H5N1) enveloped virus. In an embodiment, the antimicrobialresidual self-sanitizing film kills at least 95% (e.g., at least 96%, atleast 97%, at least 98%, at least 99%) of a log 4 population ofinfluenza A (H1N1) (ATCC CCL-34) enveloped virus within 60 minutes(e.g., within 45 minutes, within 30 minutes, within 20 minutes) ofcontact. In a preferred embodiment, the antimicrobial residualself-sanitizing film kills at least 99% of a log 4 population ofinfluenza A (H1N1) (ATCC CCL-34) enveloped virus within 30 minutes ofcontact of contact.

In yet another aspect of the invention, an antimicrobial residualself-sanitizing film renders the surface virucidal against anon-enveloped virus, such as levivirus (e.g., MS2), norovirus,rotavirus, adenovirus, parvovirus, or poliovirus. In an embodiment, theantimicrobial residual self-sanitizing film kills at least 95% (e.g., atleast 96%, at least 97%, at least 98%, at least 99%) of a non-envelopedvirus within 30 minutes of contact (e.g., within 20 minutes, within 15minutes, within 10 minutes, within 5 minutes) of contact. In a preferredembodiment, the antimicrobial residual self-sanitizing film kills atleast 97% of a non-enveloped virus within 5 minutes of contact. In someinstances of this embodiment, the non-enveloped virus is MS2 (ATCC15597-B1).

One embodiment of the invention is directed to treated filter media thatcomprises one or more insoluble cationic polymers, as described herein,that is coupled to positively charged non-woven filter media. The filtermedia is suitable for filtering, for example, liquids (e.g., water) andair and can be made from any suitable material, such as alumina (Al₂O₃),polyester (e.g., PET), polyethylene, polypropylene, polyamide (e.g.,nylon 6,6), polyimide, polyacrylic, glass, metal, dextran, cellulose,jute, wood pulp, cotton, or a combination thereof (e.g., microglassfibers and/or cellulose coated with nanoalumina fibers). If the materialis not positively charged in its natural form, the material can bemodified, as needed, to provide the necessary positive charge, by forexample, adding one or more quaternary ammonium groups. The non-wovenfilter media can be purchased commercially and can be prepared by anysuitable method (e.g., wetlaid, airlaid, drylaid, meltblown, spunbond,nanofiber web spinning, and continuous draw fiberization). See, e.g.,Argonide (Sanford, Fla.), Pall Corporation (Port Washington, N.Y.), GEInfrastructure Water and Process Technologies (Trevose, Pa.), andMeissner Filtration Products (Camarillo, Calif.). An adhesion promoterthat acts as a coupling agent, as described herein, can be used.Embodiments in which the adhesion promoter is cationic, such as branchedcarboxolated PEI, are preferred.

Most filtration media reduce pathogenic microbes by simple screening bysize, but such filters require high pressure to effective screen amaterial (e.g., fluid), easily foul, and require frequent maintenance. Atreated filter that is positively charged and coupled to one or moreinsoluble cationic polymers, as described herein, can effectively killmicroorganisms with reduced pressure and/or less fouling. FIG. 2Aillustrates a small pore size from a filter comprising 5 μm glass thatis not positively charged. FIG. 2B illustrates a filter comprisingpositively charged alumina with a larger pore size. However, the filteracts like the smaller pore sized microglass filter in FIG. 2A, becauseof the cationic polymer (e.g., insoluble polyDADMAC, linear PEI) that iscoupled to the alumina.

In one example of a treated water filter, polyDADMAC that has been madeinsoluble (e.g., by substituting part of the chloride counterions withfluoride) is coupled to positively charged, non-woven filter Al₂O₃ mediawith branched carboxylated PEI. The resulting treated filter media has avery high positive zeta value. In another example, a treated air filteris made by coupling linear PEI to positively charged, non-woven filterAl₂O₃ media using an adhesion promoter, such as branched carboxylatedPEI.

Upon testing, a highly contaminated (log 7) metal working fluid passedthrough a treated filter, as described herein, reduced microbes,including a non-enveloped virus, by 99.9%.

The invention is further illustrated by the following embodiments.

(1) An antimicrobial composition comprising: (a) a cationic polymer, (b)at least one adhesion promoter, (c) optionally organic and/or inorganicparticles that are photocatalytically active in visible light, and (d) acarrier, wherein the components of the composition are not covalentlybound to one another, and the antimicrobial composition is in accordancewith one or more of the following tests: (i) a germicidal spray testaccording to American Society for Testing and Materials (ASTM)international method E1153 that meets the EPA requirement of log 3reduction for viruses and a log 5 reduction for bacteria, (ii) asuspension test according to ASTM international method E1052-96 (2002)or ASTM international method E2315 (2016), (iii) a film formed from thecomposition kills (iii-a) at least 95% of log 5 population of a grampositive or gram negative bacteria in 30 minutes, (iii-b) at least 95%of log 4 population of an enveloped virus within 30 minutes of contactof contact, (iii-c) at least 95% of a non-enveloped virus within 30minutes of contact, (iii-d) at least 94% of a log 4 population ofClostridium difficile bacteria within 24 hours of contact, in accordancewith Japanese Industrial Standard (JIS) Z 2801 (2006) test forantimicrobial activity, or a modified version of such test as describedherein, (iv) a film formed from the composition has a value of 2 or lessaccording to International Organization for Standardization (ISO)10993-5 in vitro cytotoxicity test; and (v) a durability test selectedfrom either (v-a) a film formed from the composition kills at least99.9% of gram-positive bacteria and gram-negative bacteria according toEnvironmental Protection Agency (EPA) Protocol #01-1A residualself-sanitizing activity test, or (v-b) waiting 7 days after filmformation, a film formed from the composition kills at least 95% ofgram-positive bacteria and gram-negative bacteria, or enveloped andnon-enveloped viruses according a modified version of Protocol #01-1Aresidual self-sanitizing activity test, as described herein.

(2) The antimicrobial composition of embodiment (1), wherein thecationic polymer is a polydiallyldialkylammonium salt, anacryloxyalkyltrialkylammonium salt, a vinylphenalkyltrialkylammoniumsalt, an acrylamidoalkyltrialkylammonium salt, apoly(acrylamide-co-diallyldialkylammonium salt), apolyethylenimine-based polymer, chitosan optionally used in combinationwith an anionic polymer, or a combination thereof.

(3) The antimicrobial composition of embodiment (2), wherein thepolydiallyldialkylammonium salt is a polydiallyldimethylammonium halide,and the halide is chloride, fluoride, an anion containing chloride, ananion containing fluoride, or a combination thereof.

(4) The antimicrobial composition of any one of embodiments (1)-(3),wherein the cationic polymer is non-chemically modified linearpolyethylenimine (PEI).

(5) The antimicrobial composition of any one of embodiments (1)-(4),wherein the at least one adhesion promoter is selected from a titanate,carboxylated branched or linear PEI, a silane compound, cationic blockcopolymers, and a polymer comprising at least one acyl group, carboxylicacid group, or carboxylic acid derivative, and a combination thereof.

(6) The antimicrobial composition of any one of embodiments (1)-(5),wherein the organic and/or inorganic particles that arephotocatalytically active in visible light are selected from the groupconsisting of graphene, g-C₃N₄, a transition metal oxide, a transitionmetal sulfide, a transition metal selenide, a dye sensitizer, aconjugated polymer, a noble metal, or a mixture thereof.

(7) The antimicrobial composition of any one of embodiments (1)-(6),wherein the organic and/or inorganic particles that arephotocatalytically active in visible light are W- and N-doped TiO₂particles that have been hydrolyzed under ultraviolet (UV) light.

(8) The antimicrobial composition of any one of embodiments (1)-(7),wherein the antimicrobial composition does not contain a germicidalsmall molecule compound.

(9) The antimicrobial composition of any one of embodiments (1)-(7),wherein the antimicrobial composition further comprises at least onegermicidal agent.

(10) The antimicrobial composition of any one of embodiments (1)-(9),wherein the antimicrobial composition further comprises one or morenon-electrolyte polymers.

(11) The antimicrobial composition of embodiment (10), wherein the oneor more non-electrolyte polymers comprises a polyacrylamide.

(12) A method of killing microbes on a surface comprising applying tothe surface the antimicrobial composition of any one of embodiments(1)-(11).

(13) The method of embodiment (12), wherein the carrier evaporates toleave a residual self-sanitizing film on the surface.

(14) The method of embodiment (13), wherein the residual self-sanitizingfilm renders the surface bactericidal, virucidal, and/or germicidal.

(15) The method of embodiment (13) or (14), wherein the residualself-sanitizing film kills one or more of the following: (i) at least95% of a log 5 population of gram positive methicillin-resistantStaphylococcus aureus (MRSA) bacteria within 30 minutes of contact; (ii)at least 95% of a log 5 population of gram negative Escherichia coli(ATCC 8739) bacteria within 30 minutes of contact; (iii) at least 95% ofa log 4 population of influenza A (H1N1) (ATCC CCL-34) enveloped viruswithin 60 minutes of contact; (iv) at least 95% of a non-enveloped viruswithin 30 minutes of contact of contact; and/or (v) at least 75% of alog 4 population of Clostridium difficile (ATCC 43598) bacteria within24 hours of contact of contact.

(16) The method of embodiment (15), wherein the non-enveloped virus isMS2 (ATCC 15597-B1).

(17) An antimicrobial composition comprising a polyethylenimine-basedpolymer, optionally a second cationic polymer selected from apolydiallyldialkylammonium salt, apoly(acrylamide-co-diallyldialkylammonium halide), chitosan, or acombination thereof, optionally a polyacid, optionally at least oneadhesion promoter, and a carrier.

(18) The antimicrobial composition of embodiment (17), wherein thepolyethylenimine-based polymer is linear PEI.

(19) The antimicrobial composition of embodiment (17) or (18), whereinthe composition comprises non-chemically modified linear PEI,polydiallyldimethylammonium chloride (polyDADMAC), optionally citricacid, a carboxylated branched PEI, and a water-alcohol carrier.

(20) The antimicrobial composition of any one of embodiments (17)-(19),wherein the composition comprises citric acid.

(21) A method of killing microbes on a surface comprising applying tothe surface the antimicrobial composition of any one of embodiments(17)-(20).

(22) An antimicrobial composition comprising at least one organic and/orinorganic particle that is photocatalytically active in visible light,at least one adhesion promoter, and a carrier, wherein a film formedfrom the antimicrobial composition kill microbes under the conditions ofJIS Z 2801 that has been modified by not requiring the inoculated filmto be covered and starting the test time after the inoculum dries.

(23) A method of killing microbes on a surface comprising applying tothe surface the antimicrobial composition of embodiment (22).

(24) A method of killing microbes on a surface comprising applying tothe surface an antimicrobial composition comprising high molecularweight polydiallyldialkylammonium salt and a carrier.

(25) The method of embodiment (24), wherein the antimicrobialcomposition further comprises a polyethylenimine-based polymer,chitosan, or a combination thereof.

(26) The method of embodiment (24) or (25), wherein the antimicrobialcomposition further comprises organic and/or inorganic particles thatare photocatalytically active in visible light.

(27) The method of any one of embodiments (24)-(26), wherein theantimicrobial composition does not contain a germicidal small moleculecompound.

(28) The method of any one of embodiments (24)-(27), wherein the carrierevaporates to leave a residual self-sanitizing film on the surface.

(29) The method of embodiment (28), wherein the residual self-sanitizingfilm kills one or more of the following: (i) at least 95% of a log 5population of gram positive methicillin-resistant Staphylococcus aureus(MRSA) bacteria within 30 minutes of contact; (ii) at least 95% of a log5 population of gram negative Escherichia coli (ATCC 8739) bacteriawithin 30 minutes of contact; (iii) at least 95% of a log 4 populationof influenza A (H1N1) (ATCC CCL-34) enveloped virus within 60 minutes ofcontact; (iv) at least 95% of a non-enveloped virus within 30 minutes ofcontact of contact; and/or (v) at least 75% of a log 4 population ofClostridium difficile (ATCC 43598) bacteria within 24 hours of contactof contact.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLES

The antimicrobial compositions for the following examples were preparedaccording to the following general procedure: (1) a highly dilutemixture of one or more cationic polymers is prepared, (2) photocatalystparticles are added as a percent weight based on cationic monomers (%wbcm), (3) a highly dilute mixture of one or more anionic polymers isprepared, (4) the dilute cationic polymer and the dilute anionicpolymers are blended to create a PEC, (5) if used, a titanate adhesionpromoter is added as a percent weight based on total monomers (% wbtm),(6) the cationic/anionic PEC is condensed (i.e., the solvent ispartially evaporated) to obtain the desired concentration used indetermining, for example, film thickness and film durability, and (7)the antimicrobial composition is further diluted for desiredmodifications. Steps 2-7 are optional depending on the desireddisinfecting composition and concentration.

Example 1

This example demonstrates the preparation of an antimicrobialcomposition in an embodiment of the invention.

The individual components and their relative amounts for forming apolyDADMAC/PEI/PAAS PEC are tabulated and provided in Table 8. Theamounts of the individual components are listed in addition to acalculation of the concentration (ppm) of the solution.

TABLE 8 Cationic Charged Solids Anionic % of monomer Solids H₂O in H₂OSolids in total Ratio of Components Mass (g) % Solids (g) (g) (g) H₂O(g) fluid n⁻/n+ pDADMAC 3  40% 1.2 775.4 1.2 0.21% 0.25 PEI 0.6 100% 0.6100 0.6 0.05% PAAS 1.5  30% 0.45 293.85 0.45 g H₂O for 773.6 Total 1.80.45 Total pDADMAC mix Cationic Polymer Anionic Polymer g H₂O in bulk1.8 Titanate (% wbtm)  5% Reduction factor - 40% by volume pDADMAC g H₂Ofor PEI mix 100 Titanate (g) 0.1125 g of H₂O for PAA 292.8 Computationof ppm mix g H₂O in bulk PAA 1.05 Photocatalyst 10% 2.25 X X = 3.21k ppm(% wbcm) Total Fluid 1169.25 Photocatalyst 0.18 701.55 1000 (g)

The antimicrobial composition that creates the PEC set forth in Table 8comprises two cationic polymers (i.e., polyDADMAC and PEI), an anionicpolymer (PAAS), a titanate, TiO₂ particles (photocatalyst), and water asthe carrier. Alcohol is not required for the creation of the PEC.Following the formation of the PEC, a certain percentage of the water isreplaced with alcohol. When the composition is used as a spray ondisinfectant, the alcohol helps kill bacteria. The alcohol also helpsthe composition dry faster to form a residual self-sanitizing film. Thisreplacement of water with alcohol can range from 5% alcohol to 90%alcohol, preferably from 35% to 70%.

Example 2

This example demonstrates the future antimicrobial protection againstgram positive methicillin-resistant Staphylococcus aureus (MRSA)bacteria and gram negative Escherichia coli (ATCC 8739) bacteriaexhibited by an antimicrobial composition in accordance with anembodiment of the invention.

Disinfecting compositions were prepared comprising either 250 kDapDADMAC or ultra-high molecular weight (1,000,000 g/mol) pDADMAC, awater-methanol mixture, titanate, and functionalized TiO₂ particles asset forth in Table 9. The kill rates and times reported are afterinoculation of a 7-day-old film with the bacteria. The bacteria testingwas conducted by an independent test laboratory, BioSan Laboratories,Inc. (Warren, Mich.), and the results for the 4.8K ppm film are setforth in Table 10. A simulated EPA durability test used herein consistsof the requisite 12 alternating wet and dry wipes with a prescribedweight. Recovered organisms from each sample were measured after 5minutes. The results are set forth in Table 10, row 4.

TABLE 9 Percent for Film (ppm) 4.8k ppm 3.2k ppm Total Fluid 500 ml 750ml (80% H₂O and 20% methanol) grams of PolyDADMAC, 40% solids 6 6 Solidsin grams 2.4 2.4 grams titanate (LICA ™ 09), 5% 0.12 0.12 weight bymonomer grams of functionalized TiO₂, 10% 0.24 0.24 weight by monomer

TABLE 10 Material and/or Special Test Initial % Killed in BacteriaTested Conditions Log 5 Minutes MRSA ultra-high MW 5.93 × 10⁵ 99.83%(ATCC 33591) pDADMAC only MRSA 250,000 g/mol MW 3.25 × 10⁴  89.4% (ATCC33591) pDADMAC only Escherichia coli ultra-high MW 2.50 × 10⁵ 99.54%(ATCC 8739) pDADMAC only Escherichia coli ultra-high MW pDADMAC 3.40 ×10⁵ 99.70% (ATCC 8739) only after EPA durability test

As is apparent from the results set forth in Table 10, the ultra-highmolecular weight pDADMAC is very effective at preventing future growthof both gram-positive (MRSA) and gram-negative (E. coli) bacteria,killing greater than 99.5% in 5 minutes. In addition, these results aremeasured after a period of 7 days demonstrating that the antimicrobialresidual self-sanitizing film is continually killing at this efficientlevel. Furthermore, the ultra-high molecular weight polyDADMAC isequally effective at killing gram-negative (E. coli) bacteria after theEPA durability test. Thus, the antimicrobial residual self-sanitizingfilm does not easily wipe off of the surface.

Example 3

This example demonstrates the future antimicrobial protection againstinfluenza A (H1N1) (ATCC CCL-34) enveloped virus and MS2 (ATCC 15597-B1)non-enveloped virus exhibited by an antimicrobial composition inaccordance with an embodiment of the invention.

Disinfecting compositions comprising pDADMAC and/or PEI, a titanate, andoptionally functionalized TiO₂ in a water-methanol mixture were preparedin accordance with either Table 8, 9, or 11. The kill rates and timesreported are after inoculation of a 7-day-old film with the virus. Thevirus testing was conducted by an independent test laboratory,Antimicrobial Test Laboratories (Round Rock, Tex.), and the results setforth in Table 12.

TABLE 11 Anionic Charged Cationic Solids % of monomer Solids H₂O Solidsin in H₂O total Ratio of Components Mass (g) % Solids (g) (g) H₂O (g)(g) fluid n⁻/n+ pDADMAC 0  40% 0 0 0 0.00% 0.2625 PEI 0.8 100% 0.8300.00 0.8 0.20% PAAS 0.7  30% 0.21 100.49 0.21 g H₂O for n/a Total 0.80.21 Total pDADMAC mix Cationic Polymer Anionic Polymer g H₂O in bulk 0Titanate (% wbtm) 5% Reduction factor - 30% by volume pDADMAC g H₂O forPEI mix 300 Titanate (g) 0.0505 g of H₂O for PAA 100 Computation of ppmmix g H₂O in bulk 0.49 Photocatalyst 0% 1.01 X X = 3.6k ppm PAA (% wbcm)Total Fluid 400.49 Photocatalyst 0 280.34 1000 (g)

TABLE 12 Material and/or % % % % % Special Test Initial Killed KilledKilled Killed Killed Virus Tested Conditions Log 5 Min 10 Min 30 Min 60Min 24 Hrs influenza A ultra high MW 4.8 × 10⁴ 98.2% 99.0% (H1NI)pDADMAC/TiO₂ (Table 9) MS2 (ATCC ultra high MW 5.5 × 10⁴ 82.3% 97.8%15597-B1) pDADMAC/TiO₂ (Table 9) MS2 (ATCC PEI Only 2.4 × 10⁴ 97.4%98.3% 99.1% 15597-B1) (Table 11) MS2 (ATCC 0.33 PEI/0.66 1.9 × 10⁴ 87.5%95.0% 15597-B1) pDADMAC/TiO₂ (Table 8)

As is apparent from the results set forth in Table 12, antimicrobialcompositions comprising pDADMAC and TiO₂ lyse 98.2% of a log 4population of influenza A (H1NI) virus within 30 minutes of contact and99% within 60 minutes. In addition, antimicrobial compositionscomprising PEI kill 97.4% of a log 4 population of the non-envelopedvirus MS2 within 5 minutes, and 99% within 30 minutes. Table 12 alsodemonstrates that disinfecting compositions comprising polyDADMAC, TiO₂,and PEI become more antiviral, particularly against non-enveloped MS2,with the addition of 33% PEI. Without PEI, 82.3% are killed within 30minutes, but with 33% PEI 95% are killed within 30 minutes. Furthermore,Table 12 demonstrates that an antimicrobial composition comprisingpDADMAC and TiO₂ only kills 82.3% of the non-enveloped MS2 virus within30 minutes of contact, this increased to 97.8% after 24 hours.

Example 4

This example demonstrates the future antimicrobial protection againstspore generating Clostridium difficile (ATCC 43598) bacteria exhibitedby an antimicrobial composition in accordance with an embodiment of theinvention.

An antimicrobial composition was prepared comprising ultra-highmolecular weight pDADMAC, a titanate, and functionalized TiO₂ in awater-methanol mixture as set forth in Table 9. The kill rates and timesreported are after inoculation of a 7-day-old film with the bacteria.The bacteria testing was conducted by an independent test laboratory,Antimicrobial Test Laboratories (Round Rock, Tex.), and the results setforth in Table 13.

TABLE 13 Material and/or % Killed C. diff Tested Special Test ConditionsInitial Log in 8 hr ATC #43598 ultra-high MW pDADMAC/TiO₂ 6.75 × 10⁵ 98%

As is apparent from the results set forth in Table 13, an antimicrobialcomposition comprising ultra-high molecular weight pDADMAC and TiO₂kills 98% of a log 5 population of Clostridium difficile (ATCC 43598)bacteria in 8 hours.

Example 5

This example demonstrates the future antimicrobial protection againstAspergillas brasliensis fungus exhibited by an antimicrobial compositionin accordance with an embodiment of the invention.

An antimicrobial composition comprising ultra-high molecular weightpDADMAC, a titanate, and functionalized TiO₂ in a water-methanol mixturewas prepared using the formulation set forth in Table 9. The kill ratesand times reported are after inoculation of a 7-day-old film with thefungus. The fungus testing was conducted by independent test laboratory,BioSan Laboratories, Inc. (Warren, Mich.) and the results set forth inTable 14.

TABLE 14 Material and/or % Killed Fungus Tested Special Test ConditionsInitial Log in 8 hr Aspergillas ultra-high MW 2.15 × 10⁴ 86% brasliensispDADMAC/TiO₂

As is apparent from the results set forth in Table 14, an antimicrobialcomposition comprising ultra-high molecular weight pDADMAC, a titanate,and TiO₂ kills 86% of a log 4 population of Aspergillas brasliensisfungus in 8 hours.

Example 6

This example demonstrates the future antimicrobial protection againstgram positive methicillin-resistant Staphylococcus aureus (MRSA)bacteria exhibited by an antimicrobial composition in accordance with anembodiment of the invention.

Disinfecting compositions were prepared in accordance with thecomponents set forth in Table 9 except that no titanium dioxide waspresent. A simulated EPA durability test used herein consists of therequisite 12 alternating wet and dry wipes with a prescribed weight.Recovered organisms from each sample were measured after 5 minutes. Theresults are set forth in Table 15.

TABLE 15 Methicillin-resistant Staphylococcus aureus (MRSA) %Antimicrobial Sample ATCC 33591 cfu/sample* Reduction Activity Untreatedcontrol 1.19 × 10⁴ n/a n/a 3.2k ppm <10 >99.92% >3.08 4.8k ppm<10 >99.92% >3.08 *cfu/sample—colony forming units per sample recovered

This example demonstrates the “kill later” antimicrobial protectionagainst MRSA exhibited by a film formed by an antimicrobial compositioncontaining polyDADMAC, a titanate, and a carrier.

Example 7

This example demonstrates the antimicrobial activity exhibited by acomposition comprising pDADMAC and a carrier.

Antimicrobial compositions were prepared comprising either low molecularweight polyDADMAC (250,000 g/mol) or ultra-high molecular weight(1,000,000 g/mol) polyDADMAC in a water-methanol (80/20) mixture. Thecomposition was coated onto a clear glass slide that was allowed to dryto form a film. The killing power of the polyDADMAC film was testedagainst Methicillin-resistant Staphylococcus aureus (MRSA). Recoveredorganisms from each sample were measured after 5 minutes. The resultsare set forth in Table 16.

TABLE 16 Molecular methicillin-resistant Weight Staphylococcus aureus(MRSA) % Antimicrobial (g/mol) Sample ATCC 33591 cfu/sample* ReductionActivity Low Untreated control 1.95 × 10⁷ n/a n/a   (250,000) 9690 1.64× 10⁵   99.16% 2.08 Ultra-High Untreated control 8.88 × 10⁶ n/a n/a(1,000,000) 9855  1.5 × 10¹ >99.99% 5.77 *cfu/sample—colony formingunits per sample recovered

It was unexpectedly discovered that a film created with an ultra-highmolecular weight polyDADMAC is considerably more effective than a lowermolecular weight (250,000 g/mol) at killing gram-positive andgram-negative bacteria. As seen in Table 16, after 5 minutes of exposureto a log 7 MRSA population, the 250,000 g/mol MW film only provides anantimicrobial activity of 2.08. In comparison, a 1,000,000 g/mol MW filmcreated an antimicrobial activity of 5.7, i.e., more than double, usingthe same amount of polymer in each case. It is believed that thedifference in the kill rate of the lower molecular weight polyDADMACcompared to the higher molecular weight polyDADMAC is likely due to thedifferences in film formation rather than differences in charge density.

Example 8

This example demonstrates providing a residual self-sanitizing film on atextile with an antimicrobial composition in an embodiment of theinvention.

An antimicrobial composition in the form of a PEC comprising 6,000 ppmpDADMAC, 1,500 ppm polyacrylic acid, 400 ppm titanate, and 0.1% w/wfunctionalized TiO₂ particles was prepared. The composition was appliedin a rinse cycle to a cloth and then was tested for antimicrobialresistance using the American Association of Textile Chemists andColorists (AATCC) test method 100, which is designed to assess theperformance of antimicrobial finishes on textiles. The test demonstratedthat the polymer-based composition was able to lysis 99.58% of a log 4MRSA population on a cloth after 4 hours (Table 17). While AATCC doesnot specify a standard, a similar test method, ISO 20743, recommends a2-Log₁₀ or 99% reduction.

TABLE 17 Percent Log₁₀ Reduction Reduction Compared to Compared to TestContact Carrier LUX control at Control at Microorganism Time TypeExposure CFU/Carrier Time Zero Time Zero S. aureus Time zero Microchemn/a 3.95E+04 n/a n/a ATCC 33591 control (MRSA) 4 hours Control ~1500 lux5.70E+04 0 wash 1.65E+02 99.58% 2.38 cycles

A second antimicrobial composition comprising 4000 ppm PEI, 2000 ppmpoly(acrylamide-co-diallyldimethylammonium chloride), and 25 ppmcarboxylated branched PEI in a carrier was prepared and had a pH ofabout 6. The composition was applied in a rinse cycle to a cloth andthen tested for antimicrobial resistance under the same conditionsabove. The results are summarized in Table 18.

TABLE 18 Percent Log₁₀ Reduction Reduction Compared Compared to controlto Control Test Micro- Contact CFU/ at Time at Time organism CarrierType Time Carrier Zero Zero S. aureus Microchem Time 4.10E+05 n/a n/aATCC 33592 control zero (MRSA) Anti- 10 min 9.10E+04 77.8% 0.65microbial 20 min 6.15E+03 98.5% 1.82 composition 30 min 3.11E+03 99.2%2.12

Example 9

This example demonstrates the antimicrobial protection against E. coliexhibited by an antimicrobial composition containing titanate.

A composition comprising titanate in water was applied to a glass slide.The coated slide was allowed to set for 5 days, and then the slide wasinoculated with a log 6 E. coli population. The pure titanate filmresulted in an 88.72% kill after 24 hours, as seen in Table 19.

TABLE 19 Escherichia coli % Antimicrobial Sample ATCC 8739 cfu/sample*Reduction Activity Untreated control >5.08 × 10⁶   n/a n/a 9853 5.73 ×10⁵ >88.72% >0.95 *cfu/sample—colony forming units per sample recovered

Example 10

This example demonstrates the antimicrobial activity of a hand sanitizercomposition in an embodiment of the invention.

At room temperature, 4000 ppm of linear PEI in water was stirredvigorously to create a PEI dispersion. Under vigorous stirring, the PEIin the dispersion was then protonated with citric acid, thereby bringingthe pH to 6 and resulting in a clear liquid. The clear liquid was thenbrought to a temperature of 70° C. Ethanol and 1,2-propanediol were thendrizzled in so as to maintain the temperature of the clear liquid at 65°C. The clear mixture was taken off the heat to avoid excessive alcoholevaporation and then stirred for a minimum of 4 hours while covered. Theresulting miscible blend comprised 4000 pm non-chemically modified,linear PEI, 72% ethanol, 5% 1,2-propanediol, 0.25% by weight citricacid, and the balance water.

The activity of the hand sanitizer composition against non-envelopedviruses was in accordance with ASTM E 1052-96 (2002) (“Standard TestMethod to Assess the Activity of Microbicides against Viruses inSuspension”). Using this test the hand sanitizer formulation inactivatedMS2 (a surrogate for non-enveloped viruses) with a 99.9% (log 3)reduction within 60 seconds of contact. The activity of the handsanitizer composition against MRSA (gram positive bacteria) and E. coli(gram negative bacteria) was in accordance with ASTM E 2315. The handsanitizer composition inactivated both bacteria with a 99.999% (log 5)reduction within 30 seconds of contact. The results of these tests aresummarized in Table 20.

TABLE 20 Percent Killed Within Percent Killed Within Pathogen SuspensionTest 30 Seconds of Contact 60 Seconds of Contact MS2 (surrogate for ASTME2315  99.7%  99.9% non-enveloped virus) MRSA ASTM E-1052-96 99.999%99.999% E. Coli ASTM E-1052-96 99.999% 99.999%

Example 11

This example demonstrates the synthesis of functionalized TiO₂ particlesin an embodiment of the invention.

TiO₂ particles were functionalized using the following method. Startingwith 1 g of tungsten-doped, 20 nm liquid-synthesized TiO₂, 5 g urea wasadded, and the mixture was calcined for 40 min at 400° C. to yieldNTiO₂. The NTiO₂ was then ground to a fine powder, to which was added 10g of milling balls for every gram of NTiO₂ plus 10% urea. The mixturewas milled for 30 min at 300 rpm. After 30 min, 200 mL of water wasadded, and the mixture was milled an additional 5 min. The milledmixture was then subjected to 160 W UV light. After 1 hour, the mixturewas decanted and centrifuged and 0.5 mM dye was added in the dark. Themixture was again decanted and centrifuged, after which water was addedonce more.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. An antimicrobial composition consisting ofcationic, non-chemically modified, linear polyethylenimine (PEI), apolydiallyldialkylammonium salt, a carrier selected from ethanol,n-propanol, iso-propanol, n-butanol, sec-butanol, and t-butanol, water,and a combination thereof, and optionally one or more componentsselected from the group consisting of (a) an adhesion promoter, (b) anon-electrolyte polymer selected from the group consisting of apolyacrylamide, a polyamine, a polyamidoamine, polyethylene glycol,polyvinyl pyrrolidone, polyvinyl alcohol, a polyacrylate, and anycombination thereof, (c) a polyacid selected from the group consistingof isocitric acid, aconitic acid, propane-1,2,3-tricarboxylic acid,hemimelitic acid, trimellitic acid, trimesic acid, prehnitic acid,meallanophanic acid, pyromellitic aicd, benzenepentacarboxylic acid,mellitic acid, ethylenediamine-N,N′-dimalonic acid (EDDM),2,2′-azanediyldisuccinic acid, 2,2′-oxydisuccinic acid (ODS),ethylenediaminedisuccinic acid (EDDS), diethylenetriaminepentaaceticacid (DTPA), ethylene diamine tetraacetic acid (EDTA),2,2′4((1,2-dicarboxyethyl)azanediyl)bis(ethane-2,1-diyl))bis(oxy))disuccinicacid, and a combination thereof, (d) a proton donor selected from thegroup consisting of nitric acid, phosphoric acid, phosphonic acid, boricacid, sulfuric acid, adipic acid, benzene 1,3,5 tricarboxylic acid,chlorosuccinic acid, cholinechloride, cis-aconitic acid, citramalicacid, cyclobutane 1,1,3,3 tetracarboxylic acid, cyclohexane 1,2,4,5tetracarboxylic acid, cyclopentane 1,2,3,4 tetracarboxylic acid,diglycolic acid, fumaric acid, glutamic acid, glutaric acid, glyoxylicacid, isocitric acid, ketomalonic acid, maleic acid, malic acid, malonicacid, nitrilotriacetic acid, oxalacetic acid, oxalic acid, phytic acid,p-toluenesulfonic acid, succinic acid, tartaric acid, tartronic acid,tetrahydrofuran 2,3,4,5 tetracarboxylic acid, tricarballylic acid,versene acids, 3-hydroxyglutaric acid, 2-hydroxypropane, 1,3dicarboxylic acid, glyceric acid, furan 2,5 dicarboxylic acid,3,4-dihydroxyfuran-2,5 dicarboxylic acid,3,4-dihydroxytetrahydrofuran-2,5-dicarboxylic acid, 2-oxo-glutaric acid,dl-glyceric acid, 2,5 furan-dicarboxylic acid, and a combinationthereof, (e) propylene glycol, (f) a thickening agent, (g) a humectant,(h) an essential oil, (i) a fruit extract, (j) a fragrance, and (k) adye, wherein the antimicrobial composition is in accordance with one ormore of the following tests: (i) a germicidal spray test according toAmerican Society for Testing and Materials (ASTM) international methodE1153 that meets the EPA requirement of log 3 reduction for viruses anda log 5 reduction for bacteria, (ii) a film formed from the compositionkills (ii-a) at least 95% of log 5 population of a gram positive or gramnegative bacteria in 30 minutes, (ii-b) at least 95% of log 4 populationof an enveloped virus within 30 minutes of contact of contact, (ii-c) atleast 95% of a non-enveloped virus within 30 minutes of contact, and/or(ii-d) at least 94% of a log 4 population of Clostridium difficilebacteria within 24 hours of contact, in accordance with JapaneseIndustrial Standard (JIS) Z 2801 (2006) test for antimicrobial activity,or a modified version of such test, and (iii) a durability test selectedfrom either (v-a) a film formed from the composition kills at least99.9% of gram-positive bacteria and gram-negative bacteria according toEnvironmental Protection Agency (EPA) Protocol #01-1A residualself-sanitizing activity test, or (v-b) waiting 7 days after filmformation, a film formed from the composition kills at least 95% ofgram-positive bacteria and gram-negative bacteria, or enveloped andnon-enveloped viruses according to a modified version of Protocol #01-1Aresidual self-sanitizing activity test.
 2. The antimicrobial compositionof claim 1, wherein the polydiallyldialkylammonium salt is apolydiallyldialkylammonium halide, a polydiallyldialkylammonium sulfate,or polydiallyldialkylammonium phosphate.
 3. The antimicrobialcomposition of claim 1, wherein the polydiallyldialkylammonium salt ispolydiallyldimethylammonium chloride (polyDADMAC).
 4. The antimicrobialcomposition of claim 1, wherein a polyacid is present in thecomposition.
 5. The antimicrobial composition of claim 1, wherein atleast one adhesion promoter is present in the composition and isselected from a titanate, branched or linear PEI having carboxylic acidgroups, a silane compound, cationic block copolymers, and a polymercomprising at least one acyl group, carboxylic acid group, or carboxylicacid derivative, and a combination thereof.
 6. The antimicrobialcomposition of claim 5, wherein the at least one adhesion promoter isbranched PEI having carboxylic acid groups.
 7. The antimicrobialcomposition of claim 1, wherein one or more non-electrolyte polymers arepresent in the composition.
 8. The antimicrobial composition of claim 7,wherein the one or more non-electrolyte polymers comprises polyvinylpyrrolidone.
 9. The antimicrobial composition of claim 1, wherein thecomposition consists of cationic, non-chemically modified linear PEI,polyDADMAC, optionally branched PEI having carboxylic acid groups,optionally polyvinyl pyrrolidone, and the carrier is water, ethanol, ora combination thereof.
 10. A method of killing microbes on a surfacecomprising applying to the surface the antimicrobial composition ofclaim
 1. 11. The method of claim 10, wherein the carrier evaporates toleave a residual self-sanitizing film on the surface.
 12. The method ofclaim 11, wherein the surface comprises a textile.
 13. The method ofclaim 11, wherein the surface is part of a filter.
 14. The method ofclaim 10, wherein the composition consists of cationic, non-chemicallymodified, linear PEI, polydiallyldimethylammonium chloride (polyDADMAC),a carrier selected from ethanol, n-propanol, iso-propanol, n-butanol,sec-butanol, and t-butanol, water, and a combination thereof, andoptionally one or more components selected from the group consisting of(a) an adhesion promoter, (b) a non-electrolyte polymer selected fromthe group consisting of a polyacrylamide, a polyamine, a polyamidoamine,polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, apolyacrylate, and any combination thereof, (c) a polyacid selected fromthe group consisting of isocitric acid, aconitic acid,propane-1,2,3-tricarboxylic acid, hemimelitic acid, trimellitic acid,trimesic acid, prehnitic acid, meallanophanic acid, pyromellitic aicd,benzenepentacarboxylic acid, mellitic acid,ethylenediamine-N,N′-dimalonic acid (EDDM), 2,2′-azanediyldisuccinicacid, 2,2′-oxydisuccinic acid (ODS), ethylenediaminedisuccinic acid(EDDS), diethylenetriaminepentaacetic acid (DTPA), ethylene diaminetetraacetic acid (EDTA),2,2′4((1,2-dicarboxyethyl)azanediyl)bis(ethane-2,1-diyl))bis(oxy))disuccinicacid, and a combination thereof, (d) a proton donor selected from thegroup consisting of nitric acid, phosphoric acid, phosphonic acid, boricacid, sulfuric acid, adipic acid, benzene 1,3,5 tricarboxylic acid,chlorosuccinic acid, cholinechloride, cis-aconitic acid, citramalicacid, cyclobutane 1,1,3,3 tetracarboxylic acid, cyclohexane 1,2,4,5tetracarboxylic acid, cyclopentane 1,2,3,4 tetracarboxylic acid,diglycolic acid, fumaric acid, glutamic acid, glutaric acid, glyoxylicacid, isocitric acid, ketomalonic acid, maleic acid, malic acid, malonicacid, nitrilotriacetic acid, oxalacetic acid, oxalic acid, phytic acid,p-toluenesulfonic acid, succinic acid, tartaric acid, tartronic acid,tetrahydrofuran 2,3,4,5 tetracarboxylic acid, tricarballylic acid,versene acids, 3-hydroxyglutaric acid, 2-hydroxypropane, 1,3dicarboxylic acid, glyceric acid, furan 2,5 dicarboxylic acid,3,4-dihydroxyfuran-2,5 dicarboxylic acid,3,4-dihydroxytetrahydrofuran-2,5-dicarboxylic acid, 2-oxo-glutaric acid,dl-glyceric acid, 2,5 furan-dicarboxylic acid, and a combinationthereof, (e) propylene glycol, (f) a thickening agent, (g) a humectant,(h) an essential oil, (i) a fruit extract, (j) a fragrance, and (k) adye.
 15. The method of claim 10, wherein the composition consists ofcationic, non-chemically modified linear PEI, polyDADMAC, optionallybranched PEI having carboxylic acid groups, optionally polyvinylpyrrolidone, and the carrier is water, ethanol, or a combinationthereof.
 16. An antimicrobial composition comprising (a) a polymericantimicrobial agent consisting of cationic, non-chemically modified,linear polyethylenimine (PEI), and a polydiallyldialkylammonium salt,(b) optionally a non-electrolyte polymer selected from a polyacrylamide,a polyamine, a polyamidoamine, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, a polyacrylate, and any combinationthereof, (c) optionally at least one adhesion promoter, and (d) acarrier selected from ethanol, n-propanol, iso-propanol, n-butanol,sec-butanol, and t-butanol, water, and a combination thereof, whereinthe antimicrobial composition does not comprise a non-polymer germicidalcompound, an antimicrobial metal, an anionic polymer, or a polymericantimicrobial agent other than (a), and the antimicrobial composition isin accordance with one or more of the following tests: (i) a germicidalspray test according to American Society for Testing and Materials(ASTM) international method E1153 that meets the EPA requirement of log3 reduction for viruses and a log 5 reduction for bacteria, (ii) a filmformed from the composition kills (ii-a) at least 95% of log 5population of a gram positive or gram negative bacteria in 30 minutes,(ii-b) at least 95% of log 4 population of an enveloped virus within 30minutes of contact of contact, (ii-c) at least 95% of a non-envelopedvirus within 30 minutes of contact, and/or (ii-d) at least 94% of a log4 population of Clostridium difficile bacteria within 24 hours ofcontact, in accordance with Japanese Industrial Standard (JIS) Z 2801(2006) test for antimicrobial activity, or a modified version of suchtest, and (iii) a durability test selected from either (v-a) a filmformed from the composition kills at least 99.9% of gram-positivebacteria and gram-negative bacteria according to EnvironmentalProtection Agency (EPA) Protocol #01-1A residual self-sanitizingactivity test, or (v-b) waiting 7 days after film formation, a filmformed from the composition kills at least 95% of gram-positive bacteriaand gram-negative bacteria, or enveloped and non-enveloped virusesaccording to a modified version of Protocol #01-1A residualself-sanitizing activity test.
 17. The antimicrobial composition ofclaim 16, wherein the polydiallyldialkylammonium salt ispolydiallyldimethylammonium chloride (polyDADMAC).
 18. The antimicrobialcomposition of claim 16, wherein the composition further comprises atleast one adhesion promoter selected from a titanate, branched or linearPEI having carboxylic acid groups, a silane compound, cationic blockcopolymers, and a polymer comprising at least one acyl group, carboxylicacid group, or carboxylic acid derivative, and a combination thereof.19. The antimicrobial composition of claim 16, wherein the compositionfurther comprises one or more non-electrolyte polymers.
 20. A method ofkilling microbes on a surface comprising applying to the surface theantimicrobial composition of claim
 16. 21. The antimicrobial compositionof claim 1, wherein the cationic, non-chemically modified, linearpolyethylenimine comprises iminium acetate.
 22. The antimicrobialcomposition of claim 16, wherein the cationic, non-chemically modified,linear polyethylenimine comprises iminium acetate.
 23. The antimicrobialcomposition of claim 1, wherein the carrier is ethanol or a combinationof ethanol and water.
 24. The antimicrobial composition of claim 16,wherein the carrier is ethanol or a combination of ethanol and water.25. The antimicrobial composition of claim 1, wherein the cationic,non-chemically modified, linear PEI and polydiallyldialkylammonium saltform a crystalline miscible blend in the carrier.
 26. The antimicrobialcomposition of claim 16, wherein the cationic, non-chemically modified,linear PEI and polydiallyldialkylammonium salt form a crystallinemiscible blend in the carrier.
 27. The antimicrobial composition ofclaim 1, wherein the composition has a pH between 3-7.
 28. Theantimicrobial composition of claim 16, wherein the composition has a pHbetween 3-7.